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

Abstract

Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clinically relevant gene transthyretin was observed at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.

Fig. 1.

Synthesis of Epoxide-Derived Lipidoid Library (A) Epoxide terminated alkyl chains and amine-containing monomers were used in synthesis of combinatorial library, (B) addition of epoxides to amines by efficient ring-opening enables parallel synthesis of library members

Fig. 2.

In vitro screening of lipidoid library. Lipidoids were screened in luciferase-expressing Hela-derived cell line. (A) Antifirefly luciferase siRNA was complexed with lipidoids and incubated with cells in presence of growth media. Relative firefly luciferase expression determined by comparison of detected protein levels in treated groups vs. untreated control. (B) Luciferase silencing at low doses of siRNA (s.d., n = 4)

Fig. 3.

In vivo silencing of Factor VII in mice. A) Top-performing lipidoids from in vitro screen were purified, formulated for serum stability and delivered intravenously to C57BL/6 mice. Mice received a single bolus administration of 3 mg/kg total siRNA via tail-vein injection and Factor VII levels were quantified 72 h postinjection. B–D) Dose response experiments with top three performing lipidoids from in vivo screen; C16-96 (B), C14-110 (C), and C12-200 (D). No lipidoid-related toxicity is observed as measured by body-weight loss (E). (s.d.,n = 3 or 4, * P < 0.005, **P < 0.001; t-test, single tailed)

Fig. 4.

In vivo persistence of C12-200-mediated silencing was investigated by monitoring Factor VII protein levels for a period of over 40 days. Mice we administered a single dose of either 1 or 0.1 mg/kg siRNA and blood samples were drawn at varying timepoints for quantitation of serum protein levels. (s.d.,n = 3)

Fig. 5.

Five hepatocellular gene targets were simultaneously silenced by a single injection of pooled siRNAs formulated with C12-200. Mice we administered a single dose and dosage was titrated from 0.2 and .005 mg/kg per siRNA. 72 h postinjection, liver tissue was harvested for analysis of gene transcript levels. (s.d.,n = 5).

Fig. 6.

C12-200-siRNA particles elicit cellular uptake by macropinocytosis. (A) Fluorescently labeled siRNA formulated with C12-200 was incubated with HeLa cells in the presence of labeled markers of various endocytic pathways. siRNA-containing particles colocalize with labeled dextran, a fluid phase marker known to enter cells via macropinocytosis (White arrows). (B) Fluorescence labeling of actin fibers reveals membrane ruffling (White arrows) and actin rearrangement, hallmark indicators of uptake by macropinocytosis, within 15 min of exposure of HeLa cells to C12-200-siRNA particles. (C,D) Prior exposure of cells to EIPA and Cytochalasin D, inhibitors of macropinocytosis and actin polymerization, respectively, reduce uptake of C12-200-siRNA particles in dose-dependent fashion. (s.d., n = 3, *** P < 0.005; ** P < 0.01; * P < 0.05

Fig. 7.

Efficacy of C12-200 in nonhuman primates. Cynomolgus monkeys (n = 3 per group) received either PBS or 0.03, 0.1, or 0.3 mg/kg siTTR formulated in C12-200 as 15 min intravenous infusions (5 mL/kg) via the cephalic vein. Liver biopsies were collected from animals at 48 h postadministration. TTR mRNA levels relative to GAPDH mRNA levels were determined in liver samples. Data points represent group mean ± s.d