Follistatin Gene Therapy for Sporadic Inclusion Body Myositis Improves Functional Outcomes

Abstract

Sporadic inclusion body myositis, a variant of inflammatory myopathy, has features distinct from polymyositis/dermatomyositis. The disease affects men more than women, most commonly after age 50. Clinical features include weakness of the quadriceps, finger flexors, ankle dorsiflexors, and dysphagia. The distribution of weakness is similar to Becker muscular dystrophy, where we previously reported improvement following intramuscular injection of an isoform of follistatin (FS344) by AAV1. For this clinical trial, rAAV1.CMV.huFS344, 6 × 10¹¹ vg/kg, was delivered to the quadriceps muscles of both legs of six sporadic inclusion body myositis subjects. The primary outcome for this trial was distance traveled for the 6-min walk test. The protocol included an exercise regimen for each participant. Performance, annualized to a median 1-year change, improved +56.0 m/year for treated subjects compared to a decline of -25.8 m/year (p = 0.01) in untreated subjects (n = 8), matched for age, gender, and baseline measures. Four of the six treated subjects showed increases ranging from 58-153 m, whereas two were minimally improved (5-23 m). Treatment effects included decreased fibrosis and improved regeneration. These findings show promise for follistatin gene therapy for mild to moderately affected, ambulatory sporadic inclusion body myositis patients. More advanced disease with discernible muscle loss poses challenges.

Keywords: 6-min walk distance; 6-min walk test; Follistatin; adeno-associated virus; gene therapy; sporadic inclusion body myositis.

Figure 1

Annualized 6MWT Change in Treated versus Untreated sIBM Groups Shown is a comparison of the median annualized change in the distance walked in the 6MWT over 1 year following rAAV1.CMV.huFS344 treatment of sIBM (treated group, n = 6) versus the untreated sIBM control group matched for age, gender, and baseline 6MWT. Treated subjects improved +56.0 m/year, whereas untreated patients’ performance decreased by −25.8 m/year (*p = 0.01). Results per subject are shown in Table 1 and Figure S1.

Figure 2

Histopathology in Pre- and Post-follistatin Treatment Muscle Biopsies (A–D) Representative H&E-stained cross sections from the pre-follistatin (A and C) and post-follistatin (B and D) muscle biopsies for subject 2 (A and B) and subject 4 (C and D). Both subjects improved in their distance walked on the 6MWT (Table 1). Post-treatment samples show fewer small basophilic regenerating fibers, less variation in fiber size, and fewer central nuclei. (E and F) The fiber size histograms for subject 2 (E) and subject 4 (F) illustrate a decrease in small fiber subpopulation and a shift toward normalization of fiber size distribution with an increase in the number of fibers within the normal size range. (G) Quantification of multinucleated fibers as percent of total number of fibers with internal nuclei showed a significant reduction in the post-treatment samples, suggestive of normalization of the internal cytoarchitecture. *p = 0.0267 by two-tailed t test.

Figure 3

Reduced Fibrosis after Follistatin Gene Therapy (A–C) Representative cross-sections stained with picrosirius red from pre-treatment (A) and post-treatment (B) biopsies. The quantification of endomysial connective tissue in pre- and post-treatment biopsies is shown as percent fibrosis for individual patients (patients 1–4) (C). Significance (*p < 0.05) was assessed by two-tailed t test. Error bars represent mean + SEM. (D) Expression levels of fibrosis markers evaluated from quadriceps muscles: TGF-β, Col1A, and fibronectin pre- and post-gene therapy (n = 4 in each) versus normal control (n = 3). Error bars represent ± SEM (reduced but not significant because of pretreatment variability).

Figure 4

Western Blot Analysis of mTOR and AMPK Signaling before and after Gene Transfer (A–C) Representative western blot images and analysis of mTOR and AMPK signaling in pre- and post-treatment quadriceps muscles of four subjects and normal control quadriceps muscles. Quantitation of mTOR targets are shown: (A) P-4EBP1^(Thr37/46), (B) P-S6P S6P9(^{Ser235-Ser236)}, and (C) P-AMPK^(Thr172). The results in (A) and (B) show increased expression levels of the phosphorylated form of proteins normalized to actin and expressed as percent of normal muscle values. The results in (C) show reduction after treatment. Error bars represent mean + SEM, showing trends following treatment without reaching significance. n = 4 in the pre- and post-treatment groups, and n = 3 in the normal muscle control group.