Silencing Myostatin Using Cholesterol-conjugated siRNAs Induces Muscle Growth

Abstract

Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85-95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes.

Figure 1

In vitro screening of Mstn siRNA sequences. siRNA screen of 84 Mstn siRNAs in a luciferase reporter assay in Hepa1-6 cells with Lipofectamine 2000. Mstn siRNAs (10 nmol/l), along with Mstn luciferase reporter plasmid (0.6 ng/µl) were cotransfected into cells and luciferase reporter activity was measured after 48 hours post-transfection as a reflection of mRNA knockdown. The four lead siRNAs selected for testing in follow-up in vivo screens are indicated by black bars. Bars represent the mean of two biological replicas ± SD.

Figure 2

In vivo screen of Mstn-chol conjugates. Four Mstn-chol siRNAs (filled shapes), PBS (□), and Placebo-chol non-targeting control (o) were screened in CD-1 mice (n = 5/group) at a 15 mpk dose by i.v. injection. (a) Mstn mRNA expression was determined based on ▵▵Ct calculations, relative to PBS, in gastrocnemius muscle 3 days postinjection. (b) Mstn protein levels were measured in serum 3 days after dosing. ***P < 0.001 (by one-way analysis of variance).

Figure 3

In vivo dose titration and duration of Mstn mRNA knockdown by lead Mstn-chol siRNA. Mstn: (1169) siRNA was tested in CD-1 mice (n = 5/group) at 5, 15, and 50 mpk (filled shapes), in addition to PBS (□) and Placebo nontargeting control (o) (50 mpk) by i.v. injection (a–c) Mstn mRNA expression was determined based on ▵▵Ct calculations, relative to PBS, in gastrocnemius, extensor digitorum longus (EDL), and triceps muscles at day 3, 7, and 21 postinjection. (d) Serum Mstn protein levels were measured at indicated timepoints. All statistical analyses were performed relative to Placebo-chol. *P < 0.05, **P < 0.01, ***P < 0.001 (by one-way analysis of variance).

Figure 4

Long-term Myostatin knockdown leads to an increase in muscle size. Mstn (1169) siRNA (♦) and Placebo nontargeting control (o) were dosed intravenously at 50 mpk into CD-1 mice (n = 12/group). (a) Mstn mRNA levels were determined based on ▵▵Ct calculations, relative to PBS, in gastrocnemius, EDL, quadriceps, triceps, and spinotrapezius muscles at day 21 postinjection. (b) Serum Mstn protein levels. (c) Correlation graph of Mstn mRNA (average of gastrocnemius, EDL, quadriceps, triceps, and spinotrapezius muscles) and serum protein levels. (d) Leg muscle size, relative to day-1 placebo control. Maximum cross-sectional area of each hindlimb was quantitated from a series of 10 micro-CT images using a custom MATLAB and Definiens Developer XD software algorithm. (e) Gastrocnemius muscle weight of rested leg and exercised leg at day 21. (f) Images of hindlimb at day 21. (g) Body weight, relative to day-1 placebo control. (h) Body composition analysis by qNMR (EchoMRI) at day 20. (i) Heart weight measurements are shown normalized to different parameters and indicate the relative change in the specified parameter by Mstn-chol treatment compared to Placebo-chol. (j) Laminin immunofluorescent staining of a cross-section of gastrocnemius muscle. Average fiber cross-sectional area and total number of fibers were quantitated using Definiens software (n = 12/group). Quantitation of mean fiber area, size frequency distribution of muscle fibers, and mean fiber number is shown. All statistical analyses were performed relative to Placebo-chol. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (by one-way analysis of variance).

Figure 5

Long-term Myostatin knockdown leads to changes in muscle function. Mstn (1169) siRNA (♦) and Placebo non-targeting control (o) were injected intravenously at 50 mpk into CD-1 mice (n = 12/group). (a) Example of a muscle fatigue curve. Fatigue curves exhibit three stages of muscle fatigue: early fatigue, late fatigue, and a nonfatigable stage. “Early fatigue,” is represented by F_max-F₀, typically representative of type IIb fibers, which use creatine phosphate as an energy source. This stage is followed by “late fatigue” (F₀-F_min), typically representative of type IIa/x fibers, which use glycogen as an energy source. The final stage of the fatigue curve is the “non-fatigable” stage (F_min), which is indicative of type I fibers, which use fatty acids as an energy source. (b,d) Muscle fatigue curves: force (b) or specific force (d) were generated from an in situ muscle function assay performed on the plantar flexor muscle group on day 21 or day 22 (n = 9 total/group). Function parameters calculated from “Force” fatigue curve (c) or calculated from “Specific force” fatigue curve (e) indicate the relative change in the specified parameter by Mstn-chol treatment compared to Placebo-chol. Statistical analyses for the force curves and the functional parameters derived from these curves were performed using two-way analysis of variance and Bonferroni's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.