Lipid-like materials for low-dose, in vivo gene silencing

Purpose: Existing siRNA delivery systems required high doses (>1 mg/kg) for hepatic gene silencing. A more potent, well-tolerated lipid-like delivery material was needed to enable low-dose RNAi therapeutics and multi-gene silencing in vivo.

Hypothesis: A combinatorial library based on epoxide-derived lipid-like materials (“lipidoids”) can yield optimized siRNA delivery vehicles with improved potency, enabling hepatocyte gene silencing at doses orders of magnitude lower than previously reported systems.

Aims: Synthesize and screen a combinatorial epoxide-derived lipidoid library for siRNA delivery in vitro. - Evaluate top lipidoids for in vivo hepatocyte gene silencing in mice using Factor VII. - Assess dose–response, durability, tolerability, and simultaneous silencing of multiple hepatic genes. - Investigate cellular uptake mechanism and validate efficacy in nonhuman primates using transthyretin (TTR).

Delivery system: Platform: Lipidoid-siRNA nanoparticles. - Lead material: C12-200, an epoxide-derived amino-alcohol lipidoid. - Formulation: C12-200, DSPC, cholesterol, and mPEG2000-DMG at molar ratio 50:10:38.5:1.5; total lipid:siRNA ratio 7:1 (wt/wt); ~80 nm diameter; ~90% siRNA entrapment. - Payloads: siRNA against Factor VII, ApoB, PCSK9, XBP1, SORT1, TTR, or luciferase. - Targeting ligand: None; hepatocyte uptake via macropinocytosis. - Route: Intravenous tail-vein injection in mice; IV infusion in cynomolgus monkeys.

Approach: In vitro: HeLa-derived dual-luciferase reporter cells; 126 lipidoids screened at lipidoid:siRNA ratios 2.5:1, 5:1, 10:1, and 15:1; dose–response at 5–50 ng siRNA. - In vivo mice: C57BL/6 mice; Factor VII silencing after single IV injection; screening dose 3 mg/kg; dose–response down to 0.01 mg/kg; durability over 40 days; multi-gene silencing with pooled siRNAs at 0.2 mg/kg per siRNA (1 mg/kg total). - Nonhuman primates: Cynomolgus monkeys (n = 3/group); siTTR at 0.03, 0.1, or 0.3 mg/kg by 15-min IV infusion; liver biopsy at 48 h. - Controls: PBS, control siRNA, LNP01 benchmark, untreated cells.

Key methods: Luciferase reporter assay for in vitro silencing. - Chromogenic Factor VII assay in mouse serum. - Branched DNA assay for hepatic mRNA levels (Factor VII, ApoB, PCSK9, XBP1, SORT1, TTR). - Clinical chemistry for tolerability (ALT, AST, ALP, bilirubin). - Dynamic light scattering for particle size. - Fluorescence microscopy and colocalization with endocytic markers. - Macropinocytosis inhibitors (EIPA, Cytochalasin D).

Key results: C12-200 enabled Factor VII silencing at doses below 0.01 mg/kg in mice, >100-fold more potent than LNP01. - Complete Factor VII silencing occurred at 24 h; protein returned to baseline by ~20 days at 0.1 mg/kg and ~35 days at 1 mg/kg. - A single injection of pooled siRNAs with C12-200 silenced five hepatic genes simultaneously (>65% silencing for all five at 0.2 mg/kg per siRNA; 1 mg/kg total). - No significant toxicity at 1 mg/kg; clinical chemistry remained within normal ranges. - In nonhuman primates, C12-200/siTTR achieved high TTR knockdown at 0.3, 0.1, and 0.03 mg/kg. - Uptake occurred primarily via macropinocytosis, colocalizing with dextran but not transferrin or cholera toxin B; EIPA and Cytochalasin D inhibited uptake.

Interpretation: C12-200 is a highly potent lipidoid for hepatocyte siRNA delivery, enabling low-dose, durable, and multi-target gene silencing in mice and high-level TTR knockdown in nonhuman primates. This formulation may expand the therapeutic potential of RNAi for liver diseases such as TTR amyloidosis and multifactorial metabolic disorders.

Limitations: No human clinical data; validation limited to mice and a small number of nonhuman primates (n = 3 per group). - Long-term toxicity, chronic dosing, and accumulation of nondegradable lipidoid material were not fully assessed. - Uptake mechanism was studied mainly in HeLa cells, not primary hepatocytes. - No active targeting ligand; delivery relies on endogenous liver tropism. - No disease efficacy endpoint; silencing of endogenous genes served as proof of concept. - Multi-gene silencing was proof of principle, not a therapeutic combination tested for efficacy. - Biodistribution beyond liver was not comprehensively characterized.