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M U S C U L A R D Y S T R O P H Y

Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophyHirofumi Komaki,1* Tetsuya Nagata,2* Takashi Saito,2* Satoru Masuda,2 Eri Takeshita,1 Masayuki Sasaki,1 Hisateru Tachimori,3 Harumasa Nakamura,4 Yoshitsugu Aoki,2 Shin’ichi Takeda2†

Duchenne muscular dystrophy (DMD) is a lethal hereditary muscle disease caused by mutations in the gene en-coding the muscle protein dystrophin. These mutations result in a shift in the open reading frame leading to loss of the dystrophin protein. Antisense oligonucleotides (ASOs) that induce exon skipping correct this frame shift during pre-mRNA splicing and partially restore dystrophin expression in mouse and dog models. We conducted a phase 1, open-label, dose-escalation clinical trial to determine the safety, pharmacokinetics, and activity of NS-065/NCNP-01, a morpholino ASO that enables skipping of exon 53. Ten patients with DMD (6 to 16 years old), carrying mutations in the dystrophin gene whose reading frame would be restored by exon 53 skipping, were administered NS-065/NCNP-01 at doses of 1.25, 5, or 20 mg/kg weekly for 12 weeks. The primary endpoint was safety; the secondary endpoints were pharmacokinetics and successful exon skipping. No severe adverse drug reactions were observed, and no treatment discontinuation occurred. Muscle biopsy samples were taken before and after treatment and compared by reverse transcription polymerase chain reaction (RT-PCR), immunofluores-cence, and Western blotting to assess the amount of exon 53 skipping and dystrophin expression. NS-065/NCNP-01 induced exon 53 skipping in dystrophin-encoding mRNA in a dose-dependent manner and increased the dystrophin/spectrin ratio in 7 of 10 patients. Furthermore, the amount of exon skipping correlated with the maximum drug concentration in plasma (Cmax) and the area under the concentration-time curve in plasma (AUC0-t). These results indicate that NS-065/NCNP-01 has a favorable safety profile and promising pharmacokinetics warranting further study in a phase 2 clinical trial.

INTRODUCTIONDuchenne muscular dystrophy (DMD) is an X-linked, ultimately fa-tal muscle disease (1) that, according to the results of newborn screen-ing programs in Ohio, USA and Wales, UK, affects approximately 1 in 5000 male live births (2, 3). The manifestations of the disease in-clude progressive muscle weakness, cardiomyopathy, and respiratory failure, which lead to premature death. DMD is caused by abnormal dystrophin, a large structural muscle protein. Mutations in the gene encoding dystrophin (4) result in reduced sarcolemma stability, in-creased intracellular calcium ion influx, and, ultimately, degeneration of muscle fibers. Antisense oligonucleotides (ASOs) can induce exon skipping in the mutated dystrophin transcript, correcting its open reading frame by splicing out the exon adjacent to the deleted region in pre-mRNA. Studies in animal models show that ASOs can restore the expression of functional dystrophin and suppress disease progres-sion (5–9). However, although dystrophin expression has been observed in early-phase clinical trials of the ASOs eteplirsen and drisapersen in patients with DMD (10, 11) and some clinical benefits were suggested in a phase 2 clinical trial of drisapersen (12), a well-powered, double- blind, placebo-controlled phase 3 trial of drisapersen has not shown a statistically significant difference from placebo (13).

The region from exon 45 to 55 in the dystrophin gene is a dele-tion mutation hotspot and therefore is a potential target for exon skip-ping. Skipping of exon 51 could theoretically be used to treat 13% of DMD patients (14). Treatment of DMD patients with eteplirsen in-duced dystrophin production in muscles (15). Consequently, the U.S. Food and Drug Administration (FDA) recently approved eteplirsen under the accelerated pathway as the first disease-modifying exon 51–skipping ASO for DMD (16). The FDA rejected another exon 51–skipping drug, drisapersen, because of safety concerns (17). Other can-didate targets of exon skipping in the mutation hotspot are exons 45 and 53, and four clinical programs developing ASO approaches, in-cluding that described here, are in phase 1 or 2 clinical trials (18–20).

Skipping of exon 53 would be expected to treat approximately 8 to 10% of patients with DMD (14). In the Japanese population, the mu-tations amenable to exon 53 skipping reportedly occur as frequently as those in exon 51 (21). After the approval of eteplirsen, the applica-bility of exon 53 skipping was potentially reduced from 10 to 8% of dystrophin gene mutations (22) because exon 52 deletion could be ameliorated by skipping either exon 51 or 53. A direct comparison of phenotypes caused by dystrophins expressed after in-frame dele-tions of exons 51 to 52 and 52 to 53 has yet to be performed. Regard-less, an effective exon 53–skipping drug could benefit some patients with DMD.

NS-065/NCNP-01, a morpholino ASO developed through com-prehensive sequence optimization, was shown to efficiently induce exon 53 skipping in dystrophin-encoding pre-mRNA in human rhab domyosarcoma cell lines and in MyoD-transduced fibroblasts from two DMD patients carrying different mutations. Treatment with NS- 065/NCNP-01 resulted in restored expression of dystrophin. On the basis of these key preliminary results, we initiated a phase 1

1Department of Child Neurology, National Center Hospital, National Center of Neu-rology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo, Japan. 2Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neu-rology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo, Japan. 3Department of Mental Health Administration, National Institute of Mental Health, National Cen-ter of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo, Japan. 4Translational Medical Center, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo, Japan.*These authors contributed equally to this work.†Corresponding author. Email: takeda@ncnp.go.jp

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

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study to assess the safety and efficacy of NS-065/NCNP-01 for the treatment of DMD patients expected to benefit from exon 53 skipping. Here, we report the results of a phase 1 clinical trial aimed at evalu-ating NS-065/NCNP-01 safety and pharmacokinetics in 10 patients with DMD. The amount of exon 53 skipping and the restoration of dystrophin expression were also monitored in all patients.

RESULTSParticipants recruited for phase 1 clinical trialHere, 10 patients with DMD, including 7 nonambulant and 3 am-bulant patients, were intravenously administered NS-065/NCNP-01 at doses of 1.25, 5, or 20 mg/kg per week for 12 weeks. This phase 1 study did not aim to evaluate functional improvement, and we in-tended to enroll nonambulant patients; however, because of the scar-city of eligible DMD patients with the targeted deletion in the DMD gene at a single institution, the protocol allowed the inclusion of ambulant patients as well. This also explains the overall small sample size in this study. All 10 participants received the intended treatment and completed the safety evaluation. There were no obvious devia-tions from the protocol. The mean age of the participants at baseline (day 0) was 11 ± 3 years (Table 1). Exon deletion patterns were classi-fied into three groups: exons 48 to 52, 45 to 52, and 49 to 52; none of the patients had single-nucleotide polymorphisms (SNPs) in the NS-065/NCNP-01–targeted region. For all patients, the in vitro assay indicated that NS-065/NCNP-01 treatment induced the expression of dystrophin mRNA with a skipped exon 53 and, consequently, the ex-pression of dystrophin protein (which was slightly shorter than wild-type dystrophin). No obvious differences were found in the amount of exon skipping in patients with different exon deletion patterns (fig. S1).

Adverse drug reactions after treatment with NS-065/NCNP-01NS-065/NCNP-01 was well tolerated at all investigated doses, and no serious adverse events were reported. The severity of adverse events was graded according to the Common Terminology Criteria for Ad-verse Events, version 4.0 (Japanese translation), published by the Japan Clinical Oncology Group (CTCAE v4·0-JCOG). Overall, 72 incidences of adverse drug reactions were recorded; among them, 4 were classi-fied as grade 2 and 68 as grade 1 (Table 2). Notable adverse drug reac-tions observed in more than two patients in the low-dose (1.25 mg/kg), medium-dose (5 mg/kg), and high-dose (20 mg/kg) cohorts included an increase in N-acetyl--d-glucosaminidase (NAG) in nine patients (three patients on a low dose, three on a medium dose, and three on a high dose), proteinuria in eight patients (1/3/4), albuminuria in seven patients (2/2/3), anemia in seven patients (1/3/3), increased interleu-kin concentrations in five patients (2/1/2), elevated brain natriuret-ic peptide concentrations in two patients (1/1/0), increased diastolic pressure in two patients (0/2/0), increased complement component C3 in two patients (2/0/0), and increased 2-microglobulin in two pa-tients (0/0/2). Proteinuria seemed to be an artifact; however, we men-tion it as an adverse drug reaction in the clinical study report because we could not identify the underlying mechanism during the study. Grade 2 adverse drug reactions included decreased ejection fraction in one patient from the medium-dose cohort, and proteinuria and eczema in two and one patient, respectively, from the high-dose cohort. In all patients, the reported adverse drug reactions resolved either without or with only symptomatic treatment, and none of the patients discon-tinued the trial as a result of an adverse event. Antibodies reactive against human dystrophin or NS-065/NCNP-01 were not detected in the serum of any of the 10 DMD patients.

Table 1. Baseline characteristics and clinical summary of patients with DMD.

Subject ID DMD exon deletion Age (years) Weight (kg) BSA (m2)

Cumulative drug dose

during treatment (mg)

Age at loss of ambulation

(years)

Corticosteroid (prednisolone)

regimen

Other drug treatments at

study entry

Cohort 1 (1.25 mg/kg)

NS-06 48–52 12 37.8 1.247 550 10 No No

NS-05 45–52 16 36.3 1.274 520 10 14 mg daily Teprenone cimetidine

NS-02 48–52 14 48.1 1,394 720 8 No No

Cohort 2 (5 mg/kg)

NS-04 45–52 8 23.8 0.902 1,420 Ambulant 15 mg intermittent Famotidine

NS-01 48–52 10 48.8 1.349 2,940 Nonambulant* 20 mg intermittent No

NS-03 45–52 11 44.3 1.150 2,630 9 10 mg daily No

Cohort 3 (20 mg/kg)

NS-07 48–52 14 83.4 1.789 19,790 12 8 mg intermittent No

NS-09 45–52 13 26.9 0.982 9,100 10 No No

NS-08 49–52 8 39.8 1.211 6,490 Ambulant No No

NS-10 45–52 6 23.4 0.848 5,750 Ambulant 15 mg daily Calcium-l-aspartate

*Nonambulant, but age at loss of ambulation unknown. BSA, body surface area.

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Concentration of NS-065/NCNP-01 in patient plasmaMeasurements of the plasma concentrations of NS-065/NCNP-01 were taken over a 10-hour time period after the final dose of NS-065/NCNP-01. The maximum drug concentration in plasma (Cmax) and the area under the concentration-time curve from time 0 to time t for drug in plasma (AUC0-t) increased in a dose-dependent manner in the low-, medium-, and high-dose patient co-horts (Fig. 1, A and B). Pharmacokinetic characteristics of NS-065/NCNP-01 are shown in Table 3. At the initial dose, the mean Cmax was 6040 ng/ml in cohort 1 (low dose) and 70,200 ng/ml in cohort 3 (high dose). Over the entire study period, T1/2 was 1.52 to 1.84 hours. Median uri-nary excretion rates for NS-065/NCNP-01 over 24 hours were 69.1% (range, 49.8 to 96.7%) for the first dose and 85.3% (range, 63.8 to 100%) for the final dose. The cor -relation of Cmax and AUC0−t with the fi -nal dose calculated based on body weight and body surface area was high (R2 > 0.9) (Fig. 1, C to F), and dose calculation based on the body surface area improved the regression coefficient.

Exon skipping and dystrophin expression in DMD patients treated with NS-065/NCNP-01Figure 2 shows exon skipping in DMD mRNA for the low-, medium-, and high- dose patient cohorts. We defined the amount of exon skipping as an absolute increase; thus, 0% indicates that all primer- specific reverse transcription polymerase chain reaction (RT-PCR) products re-tained exon 53, whereas 100% indicates that all products had exon 53 excised. Exon skipping increased for all DMD pa-tients. Only one patient in cohort 1 dem- onstrated exon skipping of more than 1%, whereas exon skipping for all patients in cohorts 2 and 3 was higher than 1%, which is consistent with a dose-dependent increase. One patient (NS-07) exhibited a marked increase in exon skipping of 47.5% (from pretreatment 0.3% to post-treatment 47.8%). A statistically significant dose-dependent increase in exon skip-ping was revealed by the Jonckheere- Terpstra trend test (P = 0.0166) (Fig. 2A). Patient NS-07 had the highest Cmax and AUC0−t for the first and final doses and had the greatest amount of exon skipping (Fig. 2B). A strong correlation was ob-served between exon skipping and the final-dose Cmax (Fig. 2C) and AUC0−t (Fig. 2D).

The posttreatment ratio of dystrophin/spectrin measured by immunofluorescence

intensity quantification of spectrin and dystrophin in the sarcolemma was increased in several patients (P < 0.05) (Fig. 3, A to E). The most striking increase was observed for patient NS-07: at pretreatment, the ratio was close to zero, whereas at posttreatment, it was 0.174 (P < 0.0001), which was 17.6% of the ratio for normal control individ-uals set at 100%. For 7 of the 10 patients (70%), the posttreatment

Fig. 1. Pharmacokinetics of the ASO NS-065/NCNP-01. Mean plasma concentration versus time of the antisense oligonucleotide (ASO) NS-065/NCNP-01 for 10 patients with Duchenne muscular dystrophy (DMD) treated with a low (cohort 1), medium (cohort 2), or high (cohort 3) dose of the ASO: (A) after the first dose and (B) after the final dose. The data are expressed as means ± SD. (C and D) Maximum drug concentration in plasma (Cmax) versus dose by body weight (C) or body surface area (D) of the patients after the final dose. (E and F) Area under the concentration-time curve from time 0 to time t for drug in plasma (AUC0-t) versus dose by body weight (E) or body surface area (F) of the patients after the final dose.

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dystrophin/spectrin ratio was significantly higher than the pretreat-ment ratio (P < 0.05) (Fig. 3D). On the basis of the distribution of the dystrophin/spectrin ratio in normal control muscle fibers, DMD muscle fibers with a ratio higher than the first percentile of the control were considered to be dystrophin-positive (Fig. 3C). The treatment with NS-065/NCNP-01 increased the number of dystrophin-positive muscle fi-bers in two patients from cohort 3 (Fig. 3E); however, except for patient NS-07, dystrophin expression was not detected by Western blotting (Fig. 4). The posttreatment dystrophin/spectrin intensity ratio for pa-tient NS-07 was 8.1% of that observed in the normal control, and no dystrophin was detected at the pretreatment stage. Serum creatine ki-nase concentration was also used as one of the endpoints. However, no significant difference was observed among measurements within indi-vidual cohorts before and after treatment (table S1).

DISCUSSIONOur study is a first-in-human clinical trial of the morpholino ASO NS-065/NCNP-01. We administered NS-065/NCNP-01 weekly for 12 weeks to 10 patients with DMD carrying a mutated dystrophin gene whose open reading frame could be restored by skipping of exon 53. NS-065/NCNP-01 was well tolerated up to a single dose of 1668 mg and a cumulative dose of 19,790 mg, as evidenced by the absence of serious adverse events. We observed a dose-dependent increase in exon skipping, which was confirmed by RT-PCR. We did not detect antibodies against newly synthesized dystrophin or NS-065/NCNP-01. Several clinical trials focusing on dystrophin restoration investigated T cell responses to newly produced dystrophin (15, 23, 24). However, we could not perform such assays in this clinical trial. The risk of cell- mediated immunity against the newly produced dystrophin should be evaluated in further clinical studies of NS-065/NCNP-01.

New drug applications for the exon 51–skipping ASOs dris-apersen and eteplirsen have already been reviewed by the FDA. For drisapersen, the new drug application was rejected in January 2016 (17), whereas eteplirsen received accelerated approval in September 2016 (16). However, there was a considerable controversy regarding potential efficacy of eteplirsen, which was debated during the FDA review process. Although the regulatory review did not question the exon-skipping model, it highlighted the importance of the study de-sign and reliable methods for quantifying dystrophin expression (25). For a proof-of-concept study, we considered several routes of NS-065/NCNP-01 administration, including single-dose and intramuscu-lar injection. However, for comprehensive assessment of drug safety, we chose repeated dose administration by systemic intravenous in-fusion. In healthy individuals, an ASO for DMD exon skipping con-

verts the full-length in-frame mRNA to mRNA with an out-of-frame deletion, which can result in down-regulation of full-length dystrophin mRNA expression and an uninterpretable safety profile. Therefore, in contrast to other phase 1 trials in healthy participants, our ASO had to be evaluated in patients with DMD. This trial was conducted in a single center, which greatly limited the number of available patients with DMD, a rare disease. Furthermore, as our drug targeted a spe-cific exon, the choice of eligible patients was further limited to those carrying a mutant DMD gene whose reading frame could be restored by exon 53 skipping. As a result, 10 DMD patients were enrolled. As safety evaluations including drug dose escalation required three or more patients for each dose, we divided the 10 patients into three cohorts. The initial dose of 1.25 mg/kg was determined on the basis of nonclinical toxicological studies of NS-065/NCNP-01 in primates; the medium dose (5 mg/kg) was escalated to fourfold of initial dose, and the high dose (20 mg/kg) was escalated to fourfold of the medium dose. Considering the acceptable study duration for patients, allocat-able resources, and minimal time for dystrophin protein expression after systemic ASO treatment, a 12-week treatment period was chosen largely based on the eteplirsen phase 2 trial, where eteplirsen (50 mg/kg per week) administered for 12 weeks increased the amount of dystrophin- positive muscle fibers in three of four patients (15). Although an in-crease in the number of dystrophin-positive muscle fibers was observed for eteplirsen (50 mg/kg per week), the effects after the 12-week treat-ment period were not significant. We considered that the shortest treatment period for which we could expect an effect on dystrophin expression by our morpholino ASO NS-065/NCNP-01 was 12 weeks. Assuming that NS-065/NCNP-01 showed linear dose dependency, the expected proportion of dystrophin-positive patients in the 20-mg/kg cohort of our study was estimated to be 30%. Because only four pa-tients received NS-065/NCNP-01 at a dose of 20 mg/kg and the other patients received lower doses, the expected number of dystrophin- positive patients was calculated as 1.2 in a total of 10 enrolled patients. To satisfy the minimum number of patients to evaluate safety for each dose under the constraint that the total patient number was 10, we did not escalate the dose beyond 20 mg/kg, and we set dystrophin expres-sion as the secondary endpoint.

No severe drug-related adverse events were observed for any NS-065/NCNP-01 dose. The most frequently reported adverse reactions were elevated NAG, proteinuria, and anemia. For eteplirsen, pro-teinuria was a rare event over the 48 weeks of treatment (15), whereas for NS-065/NCNP-01, proteinuria was observed in 80% (8 of 10) of patients in this study. However, it was detected only in 24-hour pooled urine samples, and not in spot samples, and so may be due to possi-ble cross-reaction of NS-065/NCNP-01 with the pyrogallol red used

Table 3. Pharmacokinetics of NS-065/NCNP-01.

ParameterInitial dose Final dose

Cohort 1 (n = 3) Cohort 2 (n = 3) Cohort 3 (n = 4) Cohort 1 (n = 3) Cohort 2 (n = 3) Cohort 3 (n = 4)

Cmax (ng/ml) 6040 ± 300 21,800 ± 4,400 70,200 ± 44,900 5640 ± 2440 19,500 ± 1,400 72,800 ± 26,400

Tmax (hours) 1.00 ± 0.00 0.833 ± 0.289 0.750 ± 0.289 0.833 ± 0.289 0.667 ± 0.289 0.875 ± 0.250

AUC0-t (ng hour/ml) 8410 ± 1310 28,700 ± 3,900 98,900 ± 54,100 8410 ± 3520 27,700 ± 8,800 115,000 ± 56,000

T1/2 (hours) 1.66 ± 0.17 1.65 ± 0.20 1.84 ± 0.76 1.78 ± 0.05 1.52 ± 0.06 1.73 ± 0.76

Vd (ml/kg) 183 ± 14 200 ± 27 264 ± 68 232 ± 79 219 ± 24 224 ± 41

CLtot (ml/hour per kg) 149 ± 21 175 ± 26 239 ± 97 162 ± 58 190 ± 52 203 ± 87

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to measure urinary proteins. We did not detect proteinuria in any patient when using the Coomassie brilliant blue method to measure urinary proteins (table S2). Albuminuria was observed in seven pa-tients but was not considered an adverse event, because it was de-tected only in spot urine samples and not in 24-hour urine samples, and it therefore was not regarded as clinically significant. These re-sults suggest the absence of obvious kidney toxicity for NS-065/NCNP-01 administered at 20 mg/kg per week for 12 weeks. Grade 1 anemia was observed in seven patients belonging to all three dosing groups, and a causal relationship was not ruled out. Hemoglobin de-creased from days 1 to 7, but exhibited a recovery trend after day 14 in cohorts 1 (two patients) and 2 (all patients); hemoglobin recovery was also observed for one patient in cohort 3. Hemoglobin fluctua-tions may be partly attributed to the high volumes of blood needed for safety evaluations in this study. Other expected adverse events including inflammatory reactions, coagulopathies, and hepatic toxicity

were not observed. Therefore, the safety profile of NS-065/NCNP-01 is consistent with those of other morpholino ASOs.

NS-065/NCNP-01 pharmacokinetics was dose-dependent, with a short half-life. The Cmax was attained in less than 1 hour for co-horts 2 and 3 and in 1 hour for cohort 1. The urinary excretion rate indicated removal of 70 to 80% of the total administered drug with-in 24 hours. The kidney function of all patients was in the normal range. Noteworthy results were obtained for patient NS-07, who had the highest body weight and who was found to have been adminis-tered 42% more drug than the patient receiving the next highest dose when doses were calculated on the basis of the body surface area. This may explain why only patient NS-07 showed obvious dystrophin expression by Western blotting, which indicated a high degree of exon skipping. Kidney dysfunction has been reported to reduce the rapid renal clearance of unmodified phosphorodiamidate morpholino oligomers (26), but in our study, all participants, including patient

Fig. 2. Dystrophin mRNA expres-sion in DMD patients after NS-065/NCNP-01 treatment. (A) Increase in the amount of exon 53 skipping posttreatment measured by reverse transcription polymerase chain re-action (RT-PCR) and Experion auto-mated electrophoresis. Measurements were done in triplicate for each pa- tient. Percentage of exon skipping was calculated as (PCR product with- out exon 53)/(total primer-specific PCR products) × 100%. Welch’s t test showed a statistically significant in -crease in exon skipping for patients in cohorts 2 and 3 (P < 0.05). The Jonckheere-Terpstra test suggested a linear increase in the amount of exon skipping with the dose of NS-065/NCNP-01 (P < 0.0166). (B) Pretreat-ment and posttreatment variations in dystrophin mRNA in patients NS-05, NS-01, and NS-07 assessed by Ex-perion automated electrophoresis. Asterisks indicate PCR products with- out exon 53. (C and D) The increase in the amount of exon skipping as a function of pharmacokinetics of the final drug dose: (C) Cmax and (D) AUC0−t.

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Fig. 3. Immunofluorescence anal-ysis of dystrophin expression. (A) Sections of tibialis anterior muscle biopsies taken from a normal healthy control (NC) individual who was un-treated and patient NS-07 before and after NS-065/NCNP-01 treatment. Mus-cle sections were double-stained with dystrophin and spectrin antibodies. Images are at the same magnification and of the same size (338 × 338 m). Scale bars, 100 m. NC muscle fibers were smaller than those of DMD patients. Parameters were adjusted such that the dystrophin/spectrin intensity ratio in NC muscle sections was 1.0. MetaMorph script excluded non spe cific staining or fibrotic re-gions and iden tified the sarcolemma. (B) Dystrophin/spectrin intensity ratio in muscle biopsies of patient NS-07. The ratio of NC (25 images) was ap-proximately 1.0, whereas that for patient NS-07 during pretreatment (103 images) was ~0 but increased after drug treatment (105 images) to 0.174 (P < 0.05, Welch’s t test). (C) Per-centage of dystrophin-positive mus-cle fibers in muscle biopsy sections from patient NS-07. On the basis of the distribution of the dystrophin/spectrin intensity ratio of each fiber in the NC muscle biopsy sections (1716 fiber images), patient NS-07 muscle fibers with a ratio higher than the first percentile (0.537, red vertical line) were considered dystrophin- positive. Patient NS-07 showed an increase in dystrophin- positive mus-cle fibers from 0% (pretreatment) to 6.3% (posttreatment). (D) Changes in the dystrophin/spectrin intensity ratio after treatment of the 10 DMD patients with NS-065/NCNP-01. Values for each patient were normalized to NC values. Welch’s t test revealed a sta-tistically significant increase for seven DMD patients (P < 0.05). (E) Changes in the numbers of dystrophin-positive muscle fibers pre treatment and post-treatment for the 10 DMD patients. Jonckheere-Terpstra trend test sug-gested that increases in the number of dystrophin-positive muscle fibers were dependent on the dose of NS-065/NCNP-01 (P = 0.0443).

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NS-07, had normal kidney function. Therefore, it is more likely that the high body weight of patient NS-07 (83.4 kg at baseline) could be a factor. Steroid treatment may exacerbate weight gain and fat mass in DMD patients; the fat fraction of patient NS-07 was considered relatively high, although we did not measure lean body mass in this study. The impact of increased body weight and fat fraction on ASO activity has not been established, and our results indicate the need for further elucidation of ASO pharmacokinetic/pharmacodynamic profiles. Furthermore, the drug dose calculated on the basis of the body surface area resulted in more pronounced differences between patient NS-07 and the other patients in the study, suggesting that the NS-065/NCNP-01 blood concentration could be controlled ac-cording to the body surface area–based dosage.

A posttreatment increase in exon skipping was observed in most of the 10 patients, most notably patient NS-07, who showed a marked increase of 47.5%; an increase this large has not been reported in previous studies of systemic ASO treatment of DMD patients (11, 12, 15, 27–29). However, caution must be exercised in interpret-ing the results because overamplification of the skipped fragment in conventional RT-PCR compared to that in digital droplet PCR has been reported (30). Skipping of exon 53 resulted in a posttreatment increase in dystrophin protein in 7 of the 10 patients, especially in patient NS-07, as indicated by the dystrophin/spectrin ratio based on immunofluorescence analysis. This ratio, calculated as a percentage

relative to the ratio for normal control individuals set at 100%, was used as a rel- ative indicator of dystrophin expression. However, we used a single normal control sample and antibodies directed against only one (C-terminal) dystrophin domain to detect dystrophin, which we did not validate for specificity. Therefore, the re- sults regarding dystrophin expression are very preliminary and must be verified using samples from more than one healthy individual and antibodies raised against different dystrophin domains (N-terminal, C-terminal, and the rod domain). This is a clear limitation of our study.

In contrast to the dystrophin/spectrin ratio, the number of dystrophin-positive muscle fibers increased only in two pa-tients (NS-07 and NS-08). A threshold of dystrophin positivity in each slide was de-termined as the first percentile value based on the distribution of the dystrophin/spectrin ratio in a simultaneously stained normal control sample. This appeared to be a conservative approach to assess dystrophin- positive muscle fibers com-pared to visual counting, which explains the difference between the data for the dystrophin/spectrin ratio and those for dystrophin- positive muscle fibers. Al-though the analysis of the dystrophin/spectrin ratio was sensitive, we consid-ered that the observed slight increases (except for patient NS-07) were unlikely to predict clinical benefit. This view is sup-

ported by the few dystrophin- positive fibers in all but one patient, and the inability of Western blotting to detect dystrophin expression except for patient NS-07.

The discrepancy between immunofluorescence data and Western blotting results regarding dystrophin expression may be attributed to the different sensitivities of these methods under the experimental conditions of our study. Thus, immunofluorescence analysis of the pre-treatment sample from patient NS-02 revealed 0.4% dystrophin- positive revertant fibers; however, Western blotting showed no dystrophin signal. Considering these observations, it is conceivable that the sensitivity of immunofluorescence including image quantifi-cation was higher than that of Western blotting.

Our study had several notable limitations including small sample size, short treatment duration, and the absence of functional endpoints as well as the lack of full validation of the dystrophin expression as-say. Our study, however, does establish that NS-065/NCNP-01 is safe and well tolerated for 12 weeks at a dose of up to 20 mg/kg per week. We also demonstrated dose-dependent exon skipping activity of NS-065/NCNP-01, which correlated with dystrophin expression. We expect that NS-065/NCNP-01 administration at higher doses over longer periods may increase exon-skipping efficiency and enhance dystrophin expression. The approved clinical dose of eteplirsen is 30 mg/kg per week, although the FDA pointed out that a much higher dose, for example, 30 mg/kg daily, would benefit some patients who

Fig. 4. Western blotting analysis of dystrophin expression. Lysates of tibialis anterior muscle biopsies taken from a normal healthy control individual who was untreated and patient NS-07 before and after treatment with NS-065/NCNP-01. Top: Lysates were loaded in triplicate and analyzed by immunoblotting with anti-dystrophin and anti- spectrin antibodies. Bottom: Signal quantification of top panel. Measured areas are boxed in a blue rectangle: BG, background; D, dystrophin (427 kDa for normal control and 389 kDa for patient NS-07); SL, spectrin- long isoform (UniProt Q01082-1, 274 kDa); SS, short isoform (UniProt Q01082-2, 253 kDa). Myosin was visualized as background. The dystrophin/spectrin signal ratio was calculated as (D − BG)/[(SL − BG) + (SS − BG)]. The ratio of NC was set as 100%. The dystrophin/spectrin signal ratio for the pretreatment patient sample could not be calculated because dystro-phin expression could not be detected. The dystrophin/spectrin signal ratio for the posttreatment patient sample was 8.1%.

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could tolerate frequent dosing (25). For NS-065/NCNP-01, on the basis of the pharmacokinetic results and the lowest blood concentra-tion needed to restore dystrophin expression found in this study, the therapeutic dose is expected to be 40 to 80 mg/kg per week. Our col-laborative partner Nippon Shinyaku Co. Ltd. announced initiation of a phase 1/2 clinical trial in Japan in February 2016 and a phase 2 clin-ical study in the United States in March 2016 (31, 32).

In conclusion, our first-in-human study of the ASO NS-065/NCNP-01 showed that the drug had a favorable safety profile in 10 DMD patients harboring an out-of-frame deletion amenable to exon 53 skipping and exhibited promising pharmacokinetics. The next step will be a phase 2 dose-finding trial that will be required before proceeding to a pivotal clinical trial.

MATERIALS AND METHODSStudy designThis study, performed from 20 June 2013 to 12 September 2014, was designed as an investigator-initiated, single-institution, open-label, uncontrolled, phase 1 exploratory trial with the primary aim to de-termine the safety of NS-065/NCNP-01 for DMD patients (UMIN Clin-ical Trials Registry: 000010964; ClinicalTrials.gov: NCT02081625). Secondary objectives were to investigate drug pharmacokinetics, the amount of exon skipping, expression of dystrophin protein, and changes in the serum concentration of creatine kinase.

The participants in the study were ten 5- to 18-year-old Japanese boys with a genetically confirmed diagnosis of DMD, who carried a mutated DMD gene with a reading frame that could be restored by skipping of exon 53. In total, 10 DMD patients, including 7 nonam-bulant and 3 ambulant individuals, were enrolled. Because this phase 1 study did not aim to evaluate functional improvement, the patient cohort mostly consisted of nonambulant patients. However, because of the rarity of the disease, ambulant patients were also included. The Registry of Muscular Dystrophy (the Japanese registry of dystrophi-nopathy) and the Muscular Dystrophy Clinical Network (the nation-wide alliance in Japan for neuromuscular clinical research), both operated by the National Center of Neurology and Psychiatry (NCNP), facilitated patient recruitment (33, 34). No outliers were excluded in this study. The study protocol was developed in consultation with the Pharmaceuticals and Medical Devices Agency and approved by the Institutional Review Board of the NCNP. The study was conducted in accordance with good clinical practice guidelines and the Declara-tion of Helsinki. Written informed consent was obtained from parents or guardians; when possible, written informed assent was obtained from participants. As the minimal cohort size for each drug dose should not be less than 3 patients, the 10 patients were distributed into three cohorts using numbers randomly assigned to each participant by a statistician. The three cohorts received different doses of the ASO NS-065/NCNP-01: 1.25 mg/kg (n = 3, group 1), 5 mg/kg (n = 3, group 2), and 20 mg/kg (n = 4, group 3). Escalation to a higher dose was allowed if safety was confirmed for all patients in the lowest-dose cohort. Appendix S1 provides the full protocol, including additional inclusion and exclu-sion criteria, discontinuation criteria, and prohibited concomitant drugs/therapies.

Treatment with the ASO NS-065/NCNP-01NS-065/NCNP-01 was synthesized and purified by Nippon Shinyaku and supplied at 25 mg/ml in 5 ml of ampoules. The concentrated NS-065/NCNP-01 was diluted to the desired dose in 100 ml of saline and

administered by drip infusion for 1 hour once a week for 12 weeks. Each cohort was led by one patient who was administered the target dose and monitored for safety for 7 days; then the remaining patients in the cohort started weekly dosing. A safety assessment committee advised the principal investigator on dose escalation, discontinuation of the study drug, and protocol modification.

Safety assessment of NS-065/NCNP-01Safety was periodically assessed by physical examination, vital signs, elec-trocardiography, echocardiography, abdominal echo, laboratory tests, and a pulmonary function test. Signs and symptoms of DMD progres-sion were not recorded as adverse events if they were within the range of prediction, but were recorded if they were more severe than anticipated.

Drug concentrations in urine and plasmaPlasma and urinary NS-065/NCNP-01 concentrations were measured by high-performance liquid chromatography/tandem mass spectrom-etry for pharmacokinetic analysis. The standard material used for the quantification included related substances, and because it was treated as a pure compound, the concentration of NS-065/NCNP-01 was ap-proximately 10% lower than the measured value. Blood samples were collected on days 0, 7, and 77, and urine samples were collected on days 0 and 77.

After magnetic resonance imaging of the tibialis anterior muscle, tibialis anterior muscle biopsy was performed at the pretreatment (within the screening period) and posttreatment (20 ± 4 days after the last drug dose) time points; either the right or left tibialis anterior muscle was used for pretreatment biopsy, and the contralateral side was used for posttreatment biopsy. Cultured primary skin fibroblasts isolated from pretreatment muscle biopsy samples were transduced with a retrovirus coexpressing MyoD and ZsGreen1 and differentiated into myotubes as previously reported (35). Cells were then treated with 10 M NS-065/NCNP-01 and analyzed for exon 53 skipping and dystrophin expression by RT-PCR and Western blot, respectively. Genomic DNA was extracted from blood samples and screened for SNPs in the NS-065/NCNP-01–targeted DMD region.

Assessment of exon 53 skipping by RT-PCRThe exon 53 skipping level was evaluated by RT-PCR using total RNA from sliced frozen muscle. Only two primer-specific bands corre-sponding to fragments with and without exon 53 were selected, and the exon-skipping level (%) was analyzed using the Experion Auto-mated Electrophoresis Station (Bio-Rad) and calculated for each primer pair as exon 53–deleted PCR fragment/(full-length PCR fragment + exon 53–deleted PCR fragment) × 100%. The specificity of the RT-PCR products was confirmed by Sanger sequencing and gel electrophoresis.

Immunofluorescence analysis of dystrophin expressionDystrophin expression was analyzed by immunofluorescence as pre-viously reported (36) with some modifications. Briefly, muscle sec-tions from the normal control sample and patient specimens were double-stained with primary anti-spectrin and anti-dystrophin anti-bodies followed by secondary antibodies labeled with Alexa Fluor 488 (for spectrin) and Alexa Fluor 647 (for dystrophin) and observed under a confocal microscope. Although the operator was not blinded to the source of the samples (post- or pretreatment biopsies), mea-sures were taken to prevent possible bias in image selection; thus, as many fields as possible were captured using the spectrin but not the dystrophin channel. Images were taken at the same conditions, including

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laser intensity, pinhole size, detector gain, and scanning resolution, after all the parameters were optimized using the normal control sam-ple. Before fluorescence intensity quantification, a masking binary image that designated areas unsuitable for quantification, for exam-ple, those with nonspecific staining, advanced fibrotic change, or ir-regular mounting on the slide, was manually generated on the basis of only on spectrin images. Sarcolemmal region detection and fluo-rescence intensity quantification of spectrin and dystrophin were per-formed through an automatic batch process controlled by MetaMorph (Molecular Devices) script; the dystrophin/spectrin ratio was calcu-lated and used as an indicator of dystrophin expression. For prelim-inary validation of immunofluorescence analysis, we measured the dystrophin level in muscle of a patient with Becker muscular dystrophy (BMD), and it showed a patchy dystrophin expression pattern. In the setting when the dystrophin/spectrin ratio in the normal control was 1.173, the respective ratio in the BMD sample was 0.481, that is, 41.0% of the normal (fig. S2).

Western blotting analysis of dystrophin expressionWestern blotting analysis was applied to confirm dystrophin expres-sion using primary anti-dystrophin and anti-spectrin antibodies; similar to immunofluorescence analysis, dystrophin expression was quantified as the dystrophin/spectrin band intensity ratio. To evalu-ate the accuracy of the method, we mixed DMD and normal muscle lysates (9:1 ratio) and then prepared a mock BMD-like lysate con-taining 10% full-length dystrophin. Our semiquantitative analysis based on Western blotting revealed 6.0% dystrophin content in the 10% dystrophin mock lysate (fig. S3).

RT-PCR, immunofluorescence, and Western blotting are described in detail in the Supplementary Materials and Methods.

Statistical analysesBecause this study was exploratory, no statistical hypothesis was tested. The safety analysis was defined as the group comprising all subjects who received more than a single dose of the drug, and the proportion of patients who developed adverse events was calculated. For pharmacokinetic evaluation, the population included all subjects with at least one pharmacokinetic observation that could be evaluated. Pharmacokinetic parameters were derived from the concentration- time profiles using Phoenix WinNonlin version 6.3 (CERTARA). Drug activity was assessed on the basis of the amount of exon skip-ping, the percentage of dystrophin-positive muscle fibers, and the dystrophin/spectrin expression ratio. Statistical analyses were per-formed using Microsoft Excel 2010 (Microsoft), SAS 9.4 (SAS Insti-tute), and R 3.2.2 (R Foundation). Two-sided testing was used, and P ≤ 0.05 was considered significant.

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/10/437/eaan0713/DC1Appendix S1. Clinical study protocol synopsisMaterials and MethodsFig. S1. Results of the in vitro dystrophin assay.Fig. S2. Preliminary validation of immunofluorescence analysis.Fig. S3. Preliminary validation of Western blotting analysis.Table S1. Change in blood creatine kinase over time.Table S2. Urinary protein in 24-hour pooled urine samples.

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Acknowledgments: We thank A. Tamaura, M. Ohata, K. Yamaguchi, K. Fukuda, M. Suzuki, R. Shimizu, T. Mizutani, I. Nishino, K. Goto, K. Tatezawa, K. Iwasawa, Y. Hayashi, M. Kanazawa, N. Minami, and Y. Goto. Writing, assembling tables and creation of high-resolution images, copyediting, fact checking, and referencing were provided by Editage. Funding: This work was supported by an Intramural Research Grant (26-6) for Neurological and Psychiatric Disorders from the NCNP; the Clinical Research Program and the Comprehensive Research on Disability Health and Welfare Program from the Ministry of Health, Labour, and Welfare of Japan (www.mhlw.go.jp/english/index.html); the Project Promoting Clinical Trials for Development of New Drugs and Medical Devices; and the Health and Labor Sciences Research Grants for Comprehensive Research on Persons with Disabilities from the Japan Agency for Medical Research and Development (AMED). Author contributions: S.T. conceived the study and obtained the funding. H.K. was the principal investigator. H.K., T.N., T.S., and S.T. designed and managed the study with assistance from E.T. and M.S. T.N., T.S., and S.M. coordinated dystrophin quantification and programmed imaging analysis. H.T. performed statistical analysis. H.N. coordinated regulatory aspects. H.K., T.N., and T.S. wrote and Y.A. reviewed the manuscript. The first three authors, H.K., T.N., and T.S., contributed equally to the study. All authors read and approved the final version of the manuscript. Competing interests: H.K. received consulting fees from PTC Therapeutics Inc. for work unrelated to this study. S.T. received consulting fees from Nippon Shinyaku for work related to this study and also received consulting fees from TAIHO PHARMA and Daiichi-Sankyo for work unrelated to this study. S.T. and T.N. are coinventors on a patent (no. JP5363655B2, US9079934B2) owned by Nippon Shinyaku and the National Center of Neurology and Psychiatry that covers an antisense oligomer that causes skipping of exon 53 in the human dystrophin gene, and a pharmaceutical composition containing the oligomer. All the other authors declare that they have no competing interests. Data and materials availability: All data for this study have been included in the paper and in the Supplementary Materials.

Submitted 1 March 2017Accepted 17 November 2017Published 18 April 201810.1126/scitranslmed.aan0713

Citation: H. Komaki, T. Nagata, T. Saito, S. Masuda, E. Takeshita, M. Sasaki, H. Tachimori, H. Nakamura, Y. Aoki, S. Takeda, Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy. Sci. Transl. Med. 10, eaan0713 (2018).

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exon 53 in patients with Duchenne muscular dystrophySystemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of

Harumasa Nakamura, Yoshitsugu Aoki and Shin'ichi TakedaHirofumi Komaki, Tetsuya Nagata, Takashi Saito, Satoru Masuda, Eri Takeshita, Masayuki Sasaki, Hisateru Tachimori,

DOI: 10.1126/scitranslmed.aan0713, eaan0713.10Sci Transl Med

gene, suggesting that a phase 2 trial of the drug is warranted.profile and pharmacokinetics, and the authors demonstrated that it effectively skipped exon 53 in the dystrophinphase 1 clinical trial of NS-065/NCNP-01 conducted in 10 patients with DMD. The drug showed a favorable safety function and halt muscle damage by skipping exon 53 in the dystrophin gene. These authors report the results of anow developed a morpholino antisense oligonucleotide, NS-065/NCNP-01, designed to recover dystrophin

haveet al.muscle cells. This dystrophin deficiency is due to mutations in the gene encoding dystrophin. Komaki normal dystrophin, a structural protein that is indispensable for muscle cell function, causes severe damage to

Duchenne muscular dystrophy (DMD) is an inherited muscle disorder that is ultimately fatal. A deficiency inExon skipping to treat DMD

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