Effects of gene replacement therapy with resamirigene bilparvovec (AT132) on skeletal muscle pathology in X-linked myotubular myopathy: results from a substudy of the ASPIRO open-label clinical trial

Figures

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  Fig. 1. Resamirigene bilparvovec dosage level and duration of immunosuppression for the 10 participants analysed in the pathology substudy. Muscle biopsy samples were collected at baseline (predose) and at weeks 24 and 48 following treatment with resamirigene bilparvovec gene therapy at either lower dose (1.3 × 1014 vg/kg) or higher dose (3.5 × 1014 vg/kg). aPrednisolone (1 mg/kg) administered for 4 weeks followed by a 4-week taper; bPrednisolone (1 mg/kg) administered for 8 weeks followed by an 8-week taper.

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  Fig. 2. Biopsy findings in Group 1 participants. H&E and NADH-TR staining of biopsies from (a) Participant 17, (b) Participant 08, and (c) Participant 05 are shown. Participants in this group received lower-dose resamirigene bilparvovec (1.3 × 1014 vg/kg) and a short duration of immunosuppression (1 mg/kg of prednisolone for 4 weeks followed by a 4-week taper). A normal biopsy age-matched to each participant's 48-week biopsy is shown for comparison. Also shown are participant's age at the time of biopsy, serum creatine kinase (CK), quantification of serum anti-AAV antibodies and anti-myotubularin (anti-MTM1) antibodies taken at or near the time of the biopsy, the values for myotubularin protein (MTM1 protein, % of normal skeletal muscle), MTM1 RNA, and vector copy number that were obtained from the biopsy tissue, CHOP INTEND score, hours per day of ventilator dependence at each of these timepoints, and maximal inspiratory pressure (MIP). Scale bar = 50 μm.

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  Fig. 3. Biopsy findings in Group 2 participants. H&E and NADH-TR staining of biopsies from (a) Participant 20, (b) Participant 21, and (c) Participant 19 are shown. Participants in this group received lower-dose resamirigene bilparvovec (1.3 × 1014 vg/kg) and a long duration of immunosuppression (1 mg/kg of prednisolone for the first 8 weeks followed by an 8-week taper). A normal biopsy age-matched to each participant's 48-week biopsy is shown for comparison. Also shown are participant's age at the time of biopsy, serum creatine kinase (CK), quantification of serum anti-AAV antibodies and anti-myotubularin (anti-MTM1) antibodies taken at or near the time of the biopsy, the values for myotubularin protein (MTM1 protein, % of normal skeletal muscle), MTM1 RNA, and vector copy number that were obtained from the biopsy tissue, CHOP INTEND score, hours per day of ventilator dependence, and maximal inspiratory pressure (MIP) at each of these timepoints. Scale bar = 50 μm.

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  Fig. 4. Biopsy findings in Group 3 participants. H&E and NADH-TR staining of biopsies from (a) Participant 25, (b) Participant 01, (c) Participant 23, and (d) Participant 02 are shown. Participants in this group received higher-dose resamirigene bilparvovec (3.5 × 1014 vg/kg) and a long duration of immunosuppression (1 mg/kg of prednisolone for the first 8 weeks followed by an 8-week taper). Note the 48-week biopsy for Participant 9 and the 24-week biopsy for Participant 12 are not shown because they did not contain muscle tissue. A normal biopsy age-matched to each participant's 48-week biopsy is shown for comparison. Also shown are participant's age at the time of biopsy, serum creatine kinase (CK), quantification of serum anti-AAV antibodies and anti-myotubularin (anti-MTM1) antibodies taken at or near the time of the biopsy, the values for myotubularin protein (MTM1 protein, % of normal skeletal muscle), MTM1 RNA, and vector copy number that were obtained from the biopsy tissue, CHOP INTEND score, hours per day of ventilator dependence, and maximal inspiratory pressure (MIP) at each of these timepoints. For Participant 02, poor muscle bulk resulted in a lack of muscle tissue in the…

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  Fig. 5. Detailed myofibre size information. Myofibre size measurements were obtained and plotted using (a) frequency histogram and (b) cumulative probability plot methods. Due to the very large numbers of fibres sampled, all comparisons between all timepoints in a given participant demonstrated statistically significant differences at a level of p < 0.001 (nonparametric Kolmogorov–Smirnov test of distribution equality).

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  Fig. 6. Quantification of key pathological endpoints. (a) Quantification of internal nucleation (left) and organelle mislocalisation characteristic of XLMTM (right). Findings are shown for all individual participants within each group, and groups are distinguished by colour. (b) Individual participant data with respect to T-tubules, L-tubules, triads, and pathology scorea. ∗p < 0.05, ∗∗p < 0.01 (Wilcoxon ranked test with the significance level set at p < 0.05). Note that statistical comparisons were made between time points by evaluating all biopsies together at each time point, and statistics were not performed on separate groups. aXLMTM pathology score grading was assigned as follows: Grade 0, no XLMTM pathology (normal muscle or muscle with scattered atrophic fibres); Grade 1, very mild XLMTM pathology (<10% of fibres with XLMTM pathology); Grade 2, mild XLMTM pathology (11–30% of fibres with XLMTM pathology); Grade 3, moderate XLMTM pathology (31–60% of fibres with XLMTM pathology); Grade 4, moderate/severe XLMTM pathology (61–80% of fibres with XLMTM pathology); and Grade 5, severe/very severe XLMTM pathology (>81% of fibres with XLMTM pathology).

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  Fig. 7. Evaluation of inflammation in muscle biopsies. (a) CD3 staining of biopsies from study participants in which inflammation was noted at any timepoint demonstrates the relative degree of inflammation observed in individual biopsies. Any participants that are not shown did not have inflammation in any of the biopsies (Note that the 24-week biopsy for Participant 02 could not be evaluated due to lack of muscle tissue.) Scale bar = 50 μm. (b) Comparison of inflammatory markers in the 24-week muscle biopsies from Participants 17 and 08. Participant 17 showed the most inflammation overall in the study at this timepoint, despite low creatine kinase levels. Participant 08 showed very little inflammation while showing the highest creatine kinase in the study at this timepoint. Note that CD3 is a marker of T cells, CD4 is a marker of helper T (TH) cells, CD8 is a marker of cytotoxic T (TC) cells, CD20 is a marker of B cells, CD68 is a marker of macrophages, and C5b9 is a marker for the membrane attack complex of the complement cascade. Scale bar = 50 μm.

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  Fig. 8. Summary of key structural changes following treatment with resamirigene bilparvovec. Key elements of XLMTM pathology visible on light microscopy include small myofibre size, central aggregations of organelles, and internally placed nuclei (top). The earliest light microscopic improvement seen in all participants was restoration of appropriate organelle localization, in many cases without an impact on fibre size or nuclear placement (middle). By 48 weeks post-treatment, organelle localization remained appropriate and myofibre size had markedly increased. Nuclear placement was variable in post-treatment biopsies and in many cases did not improve with treatment.