Follistatin gene delivery enhances muscle growth and strength in nonhuman primates

Abstract

Antagonists of myostatin, a blood-borne negative regulator of muscle growth produced in muscle cells, have shown considerable promise for enhancing muscle mass and strength in rodent studies and could serve as potential therapeutic agents for human muscle diseases. One of the most potent of these agents, follistatin, is both safe and effective in mice, but similar tests have not been performed in nonhuman primates. To assess this important criterion for clinical translation, we tested an alternatively spliced form of human follistatin that affects skeletal muscle but that has only minimal effects on nonmuscle cells. When injected into the quadriceps of cynomolgus macaque monkeys, a follistatin isoform expressed from an adeno-associated virus serotype 1 vector, AAV1-FS344, induced pronounced and durable increases in muscle size and strength. Long-term expression of the transgene did not produce any abnormal changes in the morphology or function of key organs, indicating the safety of gene delivery by intramuscular injection of an AAV1 vector. Our results, together with the findings in mice, suggest that therapy with AAV1-FS344 may improve muscle mass and function in patients with certain degenerative muscle disorders.

Conflict of interest statement

Competing interests: J.R.M., B.K.K., and their institution (Nationwide Children’s Hospital) have filed a provisional patent on gene delivery of myostatin inhibitors and follistatin use.

Figures

Fig. 1

Injection of AAV1-FS344 into the quadriceps increases muscle mass in cynomolgus macaques. (A) AAV1-FS344 was administered by three direct unilateral injections into the right quadriceps muscle (total dose of 1 × 1013 vector genomes in 1.5 ml). Indian ink tattoos, drawn immediately after the injections, allowed the vector to be localized at necropsy. (B) Concentrations of human follistatin in muscle, as measured by enzyme-linked immunosorbent assay at 5 and 15 months after injection. The values represent individual macaques with four muscle samples analyzed per macaque. (C) Increases in quadriceps size after injection of AAV1-FS344, driven by either the CMV-FS or the MCK-FS promoter. Mean ± SEM values for three macaques per treatment are shown. Asterisks indicate a 15% increase over baseline at 8 weeks in the CMV-FS group (P = 0.01) and a 10% increase in the MCK-FS group (P = 0.02). (D) Quadriceps enlargement seen at necropsy of MCK-FS and CMV-FS macaques.

Fig. 2

AAV1-FS344 treatment causes myofiber hypertrophy and affects fast-twitch type 2 myofibers in the quadriceps muscle. (A) H&E staining of quadriceps muscle reveals myofiber hypertrophy in CMV-FS macaque (right) compared to untreated control (left). Scale bars, 20 μm. (B) Morphometric analysis of quadriceps muscle at 5 months after AAV1-FS344 injection demonstrates a significant increase in mean fiber diameter (dotted lines) of both MCK-FS (P < 0.04, n = 3 muscle samples) and CMV-FS (P < 0.001, n = 3 muscle samples) animals relative to untreated controls (n = 3 muscle samples). (C) Representative fiber types determined by ATPase staining (pH 4.6) of quadriceps muscle from CMV-FS macaques (right) and untreated controls (left). Scale bars, 20 μm. (D) Fiber type ratios in control, CMV-FS, and MCK-FS muscles. Mean ± SEM values for three animals are shown. (E) Mean ± SEM fiber diameters (dotted lines) of fast-twitch oxidative glycolytic type 2a and fast-twitch glycolytic 2b fibers show significant increases in three CMV-FS animals relative to untreated controls (P < 0.001); slow-twitch oxidative type 1 fibers are not affected. Similar but not statistically significant trends are apparent in the MCK-FS group (n = 3).

Fig. 3

The FS344 transgene and the AAV1 capsid do not produce cellular immune responses. (A) PBMCs were isolated before injection and at monthly intervals thereafter and then were stimulated with human follistatin peptide pools (I and II) and AAV1 capsid peptide pools (I, II, and III) or with concanavalin A (ConA; positive control). IFN-γ release was measured with an ELISpot assay; representative data obtained at 5 months after injection are shown. (B) Mean ± SEM SFCs for each antigen per 1 × 106 PBMCs are shown over 5 months for each treatment group (n = 3). Increases in SFC did not exceed 25, the threshold (red dashed line) for significance with this assay in our laboratory.

Fig. 4

AAV1-FS344 treatment does not cause hypertrophy of cardiomyocytes. (A) H&E staining of cardiac muscle from boththe MCK-FSand the CMV-FS groups reveals normal cardiac muscle histology compared with nontreated controls. Scale bars, 50 μm. (B) Morphometric analysis shows similar mean ± SEM diameters of cardiomyocytes from AAV1-FS344–treated macaques (MCK-FS and CMV-FS groups) compared with untreated controls (n = 3 per group).