Research ArticleMuscular Dystrophy

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

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Science Translational Medicine  18 Apr 2018:
Vol. 10, Issue 437, eaan0713
DOI: 10.1126/scitranslmed.aan0713
  • 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.

  • Fig. 2 Dystrophin mRNA expression 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 reaction (RT-PCR) and Experion automated electrophoresis. Measurements were done in triplicate for each patient. Percentage of exon skipping was calculated as (PCR product without exon 53)/(total primer-specific PCR products) × 100%. Welch’s t test showed a statistically significant increase 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) Pretreatment and posttreatment variations in dystrophin mRNA in patients NS-05, NS-01, and NS-07 assessed by Experion automated electrophoresis. Asterisks indicate PCR products without 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.

  • Fig. 3 Immunofluorescence analysis of dystrophin expression.

    (A) Sections of tibialis anterior muscle biopsies taken from a normal healthy control (NC) individual who was untreated and patient NS-07 before and after NS-065/NCNP-01 treatment. Muscle sections were double-stained with dystrophin and spectrin antibodies. Images are at the same magnification and of the same size (338 × 338 μm). Scale bars, 100 μm. 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 nonspecific staining or fibrotic regions and identified the sarcolemma. (B) Dystrophin/spectrin intensity ratio in muscle biopsies of patient NS-07. The ratio of NC (25 images) was approximately 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) Percentage of dystrophin-positive muscle 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 muscle 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 statistically significant increase for seven DMD patients (P < 0.05). (E) Changes in the numbers of dystrophin-positive muscle fibers pretreatment and posttreatment for the 10 DMD patients. Jonckheere-Terpstra trend test suggested that increases in the number of dystrophin-positive muscle fibers were dependent on the dose of NS-065/NCNP-01 (P = 0.0443).

  • 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-β 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 dystrophin expression could not be detected. The dystrophin/spectrin signal ratio for the posttreatment patient sample was 8.1%.

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

    Subject IDDMD 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-0648–521237.81.24755010NoNo
    NS-0545–521636.31.2745201014 mg dailyTeprenone
    cimetidine
    NS-0248–521448.11,3947208NoNo
    Cohort 2 (5 mg/kg)
    NS-0445–52823.80.9021,420Ambulant15 mg intermittentFamotidine
    NS-0148–521048.81.3492,940Nonambulant*20 mg intermittentNo
    NS-0345–521144.31.1502,630910 mg dailyNo
    Cohort 3 (20 mg/kg)
    NS-0748–521483.41.78919,790128 mg intermittentNo
    NS-0945–521326.90.9829,10010NoNo
    NS-0849–52839.81.2116,490AmbulantNoNo
    NS-1045–52623.40.8485,750Ambulant15 mg dailyCalcium-l-
    aspartate

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

    • Table 2 Summary of observed adverse drug reactions.
      Cohort 1 (n = 3)Cohort 2 (n = 3)Cohort 3 (n = 4)Total (n = 10)
      PatientsEventsPatientsEventsPatientsEventsPatientsEvents
      All adverse drug reactions324323 (1)425 (3)1072 (4)
      Blood and lymphatic
      system disorders
      12333378
      Anemia12333378
      Skin and subcutaneous
      tissue disorders
      000011 (1)11 (1)
      Eczema000011 (1)11 (1)
      Renal and urinary disorders11000011
      Hematuria11000011
      Laboratory and physical
      examination
      321320 (1)421 (2)1062 (3)
      Albuminuria22223377
      β-2-Microglobulin increased00002222
      N-acetyl-β-d-glucosaminidase
      increased
      353433912
      Blood fibrinogen decreased00110011
      Blood fibrinogen increased11000011
      Blood glucose increased00001111
      Diastolic pressure increased00230023
      C-reactive protein increased11000011
      Complement component
      C3 increased
      22000022
      Urinary blood present11000011
      Oxygen saturation decreased00001111
      Respiratory rate increased00001111
      White blood cell count increased11000011
      Ejection fraction decreased0011 (1)0011 (1)
      Proteinuria*123648 (2)816 (2)
      Brain natriuretic peptide increased11110022
      Urine protein/creatinine
      ratio increased
      11000011
      Urine ketone body present11000011
      Cystatin C increased00110011
      Tumor necrosis factor-α increased11000011
      Interleukin level increased22112255

      Values in parentheses include events classified as a grade 2 adverse drug reaction.

      *Results of the pyrogallol red method. Remeasurement using the Coomassie brilliant blue method showed no proteinuria.

      • Table 3 Pharmacokinetics of NS-065/NCNP-01.
        ParameterInitial doseFinal 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 ± 30021,800 ± 4,40070,200 ± 44,9005640 ± 244019,500 ± 1,40072,800 ± 26,400
        Tmax (hours)1.00 ± 0.000.833 ± 0.2890.750 ± 0.2890.833 ± 0.2890.667 ± 0.2890.875 ± 0.250
        AUC0-t (ng hour/ml)8410 ± 131028,700 ± 3,90098,900 ± 54,1008410 ± 352027,700 ± 8,800115,000 ± 56,000
        T1/2 (hours)1.66 ± 0.171.65 ± 0.201.84 ± 0.761.78 ± 0.051.52 ± 0.061.73 ± 0.76
        Vd (ml/kg)183 ± 14200 ± 27264 ± 68232 ± 79219 ± 24224 ± 41
        CLtot (ml/hour per kg)149 ± 21175 ± 26239 ± 97162 ± 58190 ± 52203 ± 87

      Supplementary Materials

      • www.sciencetranslationalmedicine.org/cgi/content/full/10/437/eaan0713/DC1

        Appendix S1. Clinical study protocol synopsis

        Materials and Methods

        Fig. 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.

      • Supplementary Material for:

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

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

        *Corresponding author. Email: takeda{at}ncnp.go.jp

        Published 18 April 2018, Sci. Transl. Med. 10, eaan0713 (2018)
        DOI: 10.1126/scitranslmed.aan07131

        This PDF file includes:

        • Appendix S1. Clinical study protocol synopsis
        • Materials and Methods
        • Fig. 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.

        [Download PDF]

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