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

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 ( \u3e 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

The chemical structure and phosphorothioate content of hydrophobically modified siRNAs impact extrahepatic distribution and efficacy

Small interfering RNAs (siRNAs) have revolutionized the treatment of liver diseases. However, robust siRNA delivery to other tissues represents a major technological need. Conjugating lipids (e.g. docosanoic acid, DCA) to siRNA supports extrahepatic delivery, but tissue accumulation and gene silencing efficacy are lower than that achieved in liver by clinical-stage compounds. The chemical structure of conjugated siRNA may significantly impact invivo efficacy, particularly in tissues with lower compound accumulation. Here, we report the first systematic evaluation of the impact of siRNA scaffold-i.e. structure, phosphorothioate (PS) content, linker composition-on DCA-conjugated siRNA delivery and efficacy in vivo. We found that structural asymmetry (e.g. 5- or 2-nt overhang) has no impact on accumulation, but is a principal factor for enhancing activity in extrahepatic tissues. Similarly, linker chemistry (cleavable versus stable) altered activity, but not accumulation. In contrast, increasing PS content enhanced accumulation of asymmetric compounds, but negatively impacted efficacy. Our findings suggest that siRNA tissue accumulation does not fully define efficacy, and that the impact of siRNA chemical structure on activity is driven by intracellular re-distribution and endosomal escape. Fine-tuning siRNA chemical structure for optimal extrahepatic efficacy is a critical next step for the progression of therapeutic RNAi applications beyond liver

Comparative route of administration studies using therapeutic siRNAs show widespread gene modulation in Dorset sheep

siRNAs comprise a class of drugs that can be programmed to silence any target gene. Chemical engineering efforts resulted in development of divalent siRNAs (di-siRNAs), which support robust and long-term efficacy in rodent and nonhuman primate brains upon direct cerebrospinal fluid (CSF) administration. Oligonucleotide distribution in the CNS is nonuniform, limiting clinical applications. The contribution of CSF infusion placement and dosing regimen on relative accumulation, specifically in the context of large animals, is not well characterized. To our knowledge, we report the first systemic, comparative study investigating the effects of 3 routes of administration - intrastriatal (i.s.), i.c.v., and intrathecal catheter to the cisterna magna (ITC) - and 2 dosing regimens - single and repetitive via an implanted reservoir device - on di-siRNA distribution and accumulation in the CNS of Dorset sheep. CSF injections (i.c.v. and ITC) resulted in similar distribution and accumulation across brain regions. Repeated dosing increased homogeneity, with greater relative deep brain accumulation. Conversely, i.s. administration supported region-specific delivery. These results suggest that dosing regimen, not CSF infusion placement, may equalize siRNA accumulation and efficacy throughout the brain. These findings inform the planning and execution of preclinical and clinical studies using siRNA therapeutics in the CNS