A phase 1/2a follistatin gene therapy trial for becker muscular dystrophy

Abstract

Becker muscular dystrophy (BMD) is a variant of dystrophin deficiency resulting from DMD gene mutations. Phenotype is variable with loss of ambulation in late teenage or late mid-life years. There is currently no treatment for this condition. In this BMD proof-of-principle clinical trial, a potent myostatin antagonist, follistatin (FS), was used to inhibit the myostatin pathway. Extensive preclinical studies, using adeno-associated virus (AAV) to deliver follistatin, demonstrated an increase in strength. For this trial, we used the alternatively spliced FS344 to avoid potential binding to off target sites. AAV1.CMV.FS344 was delivered to six BMD patients by direct bilateral intramuscular quadriceps injections. Cohort 1 included three subjects receiving 3 × 10(11) vg/kg/leg. The distance walked on the 6MWT was the primary outcome measure. Patients 01 and 02 improved 58 meters (m) and 125 m, respectively. Patient 03 showed no change. In Cohort 2, Patients 05 and 06 received 6 × 10(11) vg/kg/leg with improved 6MWT by 108 m and 29 m, whereas, Patient 04 showed no improvement. No adverse effects were encountered. Histological changes corroborated benefit showing reduced endomysial fibrosis, reduced central nucleation, more normal fiber size distribution with muscle hypertrophy, especially at high dose. The results are encouraging for treatment of dystrophin-deficient muscle diseases.

Figure 1

Distance walked in 6-minute walk test (6MWT) following follistatin gene therapy. (a) Distance walked in meters in the 6MWT for subjects receiving AAV1.CMV.FS344 in each leg (3 × 10¹¹ vg/kg/leg) with follow up for 1 year. A stippled red line shows the baseline for each patient. Patients are numbered consecutively based on treatment at ~4–6 week intervals. (b) The table shows the exact distances at each time point from baseline (BL) to 1 year. The “12-mo change” indicates the distance walked compared to BL. (c) Distance walked in meters in the 6MWT for subjects receiving AAV1.CMV.FS344 in each leg (6 × 10¹¹ vg/kg/leg) with follow up for 6 months. (d) The table again shows the exact distances at each time point from baseline (BL) to 6 months. The “6-mo change” indicates the distance walked compared to BL. D, day.

Figure 2

Interferon-gamma (IFN-γ) ELISpot assays. The T cell immune responses to AAV1 capsid and follistatin are shown for each patient throughout the clinical trial. Spot forming cells (SFCs) per million peripheral blood mononuclear cells (PBMCs) are shown on the Y-axis, and days postinfection (dpi) on X-axis.

Figure 3

Site of gene transfer on leg compared to areas of fibrosis. (a) The sites of gene transfer to the right leg is shown for Patient 05 (distance walked = 108 m, 6MWT) using a surgical marking pen; (b) MRI of quadriceps muscles for Patient 05 shows a mild degree of MRI intensity (T1-weighted image); (c) the sites of gene transfer to the right leg is shown for Patient 03 (distance walked = 9 m, 6MWT) using a surgical marking pen; (d) MRI of quadriceps muscles for Patient 03 shows marked increase in intensity indicative of fibrosis.

Figure 4

Grading Scale for quadriceps muscles by magnetic resonance images (MRI). Muscle MRIs were used to establish a grading scale for the quadriceps muscles based on approximate percentage of increased image intensity indicating degree of fibrosis replacing normal muscle. There was an overall correlation between fibrosis and distance walked on the 6MWT with Patients 03 and 04 demonstrating the least benefit from gene transfer. RF, rectus femoris; VL/VI, vastus lateralis/vastus intermedius; VM, vastus medialis.

Figure 5

Focal areas of clinical muscle hypertrophy. Following gene transfer, focal areas of muscle hypertrophy (red arrows) could be seen clinically, as shown in Patients 01 and 05. We never observed diffuse quadriceps muscle enlargement as we had seen in preclinical studies in the nonhuman primate.

Figure 6

Muscle biopsy changes following follistatin gene therapy. (a) Pretreatment biopsy from Patient 05; (b) Posttreatment biopsy from Patient 05; (c) Pretreatment biopsy from Patient 06; (d) Posttreatment biopsy from Patient 06. The posttreatment biopsies show reduced fibrosis and a decrease in central nucleation. The number of small muscle fibers is markedly reduced and fewer split fibers are seen. Fiber size analyses showed a shift toward larger mean fiber diameter populations: Patient 05, prebiopsy 40.14 ± 2.10 µm (n = 323 fibers) versus postbiopsy 59.33 ± 1.54 µm (n = 292 fibers); P < 0.0001; Patient 06, pre 47.48 ± 2.00 µm (n = 245 fibers) versus post 63.74 ± 2.45 µm (n = 277) P < 0.0001.

Figure 7

Reduced fibrosis following follistatin gene therapy. Percent fibrosis using picrosirius staining was quantified comparing pre- and posttreatment muscle biopsies in high dose cohort. The error bars represent standard error of the mean. Posttreatment, we found that fibrosis was reduced to 35% of baseline for Patient 05 and to 43% of baseline for patient 06 (P < 0.017; mean percent fibrosis in Cohort 2 pretreatment 33.14 ± 4.47 versus posttreatment 19.28 ± 1.73; one-way analysis of variance).