Docosanoic acid conjugation to siRNA enables functional and safe delivery to skeletal and cardiac muscles

Abstract

Oligonucleotide therapeutics hold promise for the treatment of muscle- and heart-related diseases. However, oligonucleotide delivery across the continuous endothelium of muscle tissue is challenging. Here, we demonstrate that docosanoic acid (DCA) conjugation of small interfering RNAs (siRNAs) enables efficient (~5% of injected dose), sustainable (>1 month), and non-toxic (no cytokine induction at 100 mg/kg) gene silencing in both skeletal and cardiac muscles after systemic injection. When designed to target myostatin (muscle growth regulation gene), siRNAs induced ~55% silencing in various muscle tissues and 80% silencing in heart, translating into a ~50% increase in muscle volume within 1 week. Our study identifies compounds for RNAi-based modulation of gene expression in skeletal and cardiac muscles, paving the way for both functional genomics studies and therapeutic gene modulation in muscle and heart.

Graphical abstract

Figure 1

DCA conjugate supports significant accumulation in muscle tissues after s.c. injection at 1 week and 1 month post-injection (A) Bar graph showing siRNA quantification after 1 week (20, 2 × 20, and 6 × 20 mg/kg) and 1 month (2 × 20 mg/kg 1 month) post-injection in liver, heart, gastrocnemius, and quadriceps, measured by PNA hybridization assay (average of n = 6 ± SD). Data analysis: t test (∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01). (B) Table summarizing percent of injected dose retained in liver, heart, and muscles (average of 6 mice ± SD).

Figure 2

Significant silencing of Mstn mRNA and MSTN protein in muscles is achieved using DCA-conjugated siRNAs (A) Percent of Mstn mRNA silencing in gastrocnemius, quadriceps, and heart after s.c. injection of DCA-conjugated siRNA in mice sacrificed at 1 week post-injection (n = 6 mice per group; 20, 2 × 20, and 6 × 20 mg/kg). mRNA levels were measured using QuantiGene (Affymetrix), normalized to a housekeeping gene, Hprt (hypoxanthine-guanine phosphoribosyl transferase), and presented as percent of Ntc (mean ± SD). (B) Percent of MSTN protein levels in serum and quadriceps. s.c. injection of DCA-conjugated siRNA in mice sacrificed at 1 week post-injection (n = 6 mice per group; 20, 2 × 20, and 6 × 20 mg/kg). Protein levels were measured using GDF-8/MSTN Quantikine ELISA kit (R&D Systems), presented as percent of PBS. Data analysis: multiple comparisons = one-way ANOVA, Dunnett test (∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.1). n.s., non-significant.

Figure 3

Long-term Mstn mRNA and protein silencing in muscles is achieved using DCA-conjugated siRNAs (A) Percent of Mstn mRNA silencing in gastrocnemius, quadriceps, and heart after s.c. injection of DCA-conjugated siRNA in mice sacrificed at 1 week and 1 month post-injection (n = 6 mice per group; 2 × 20 mg/kg). mRNA levels were measured using QuantiGene (Affymetrix), normalized to a housekeeping gene, Hprt, and presented as percent of Ntc (mean ± SD). (B) Percent of MSTN protein levels in serum and quadriceps. s.c. injection of DCA-conjugated siRNA in mice sacrificed at 1 week and 1 month post-injection (n = 6 mice per group, 2 × 20 mg/kg). Protein levels were measured using GDF-8/MSTN Quantikine ELISA kit (R&D Systems), presented as percent of PBS. Data analysis: multiple comparisons = one-way ANOVA, Dunnett test (∗∗p < 0.01).

Figure 4

Mstn silencing in muscles leads to a significant increase in muscle growth (A) Thigh diameter normalized to PBS and representative images of mouse hindlimbs after injection at various doses. (B) Body weight normalized to body weight at day −2. (C) Relative change in heart weight compared with PBS group and heart weight/body weight ratio normalized to the PBS group. s.c. injection of DCA-conjugated siRNA in mice sacrificed at 1 week and 1 month post-injection (n = 6 mice per group, mean ± SD; 20, 2 × 20, and 6 × 20 mg/kg). Data analysis: multiple comparisons = one-way ANOVA, Dunnett test and two-way ANOVA, Tukey test (∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01).

Figure 5

DCA conjugate shows a safe cytokine profile at high doses Bar graph showing cytokine levels relative to PBS. 24 h after s.c. injections of DCA- and cholesterol-conjugated siRNAs at 50 and 100 mg/kg (n = 3 mice per group, mean ± SD). Data analysis: multiple comparisons = one-way ANOVA, Dunnett test (∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.1).

Figure 6

DCA conjugate shows a safe cytokine profile after multiple injections Bar graph showing cytokine levels relative to PBS. 1 week after s.c. injections of DCA-conjugated siRNAs at 20, 2 × 20, and 6 × 20 mg/kg (n = 3 mice per group, mean ± SD). Data analysis: multiple comparisons = one-way ANOVA, Dunnett test (∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.1).