Development of lipidoid-siRNA formulations for systemic delivery to the liver

Abstract

RNA interference therapeutics afford the potential to silence target gene expression specifically, thereby blocking production of disease-causing proteins. The development of safe and effective systemic small interfering RNA (siRNA) delivery systems is of central importance to the therapeutic application of siRNA. Lipid and lipid-like materials are currently the most well-studied siRNA delivery systems for liver delivery, having been utilized in several animal models, including nonhuman primates. Here, we describe the development of a multicomponent, systemic siRNA delivery system, based on the novel lipid-like material 98N(12)-5(1). We show that in vivo delivery efficacy is affected by many parameters, including the formulation composition, nature of particle PEGylation, degree of drug loading, and biophysical parameters such as particle size. In particular, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can result in significant effects on in vivo efficacy. The lead formulation developed is liver targeted (>90% injected dose distributes to liver) and can induce fully reversible, long-duration gene silencing without loss of activity following repeat administration.

Figure 1

The structures of 98N₁₂-5(1), the five-tailed isomer of triethylenetetramine–laurylaminopropionate with a free internal amine, Cholesterol, and mPEG₂₀₀₀-C14 Glyceride.

Figure 2

Maximization of siRNA loading into 98N₁₂-5(1)-based formulation. (a) siRNA entrapment at various initial lipid:siRNA (wt:wt) ratios. (b) Removal of nonentrapped siRNA from a formulation with initial lipid:siRNA ratio of 5:1 using tangential flow filtration (TFF) (100,000 molecular weight cutoff). Entrapment was measured before and after TFF with five volumes of buffer exchange. (c,d) C57BL/6 mice (n = 3) received a single intravenous dose of 1.5, 2, or 10 mg/kg of Factor VII targeting siRNA formulated at different lipid:siRNA ratios. Control animals received saline injections. (c) Weight gain 48 hours postadministration for 10 mg/kg groups shown. (d) Serum Factor VII protein levels measured 48 hours postadministration. Data points represent group mean ± SD. PBS, phosphate-buffered saline; siRNA, small interfering RNA.

Figure 3

Impact of PEG lipid alkyl chain length and particle size on in vivo activity. (a,b) C57BL/6 mice (n = 3) received a single intravenous dose of either 2.5 or 20 mg/kg of Factor VII targeting siRNA formulated with PEG-lipids with different alkyl chain lengths (C10–C16). Control animals received saline injections. (a) Weight gain and (b) serum Factor VII protein levels 48 hours postadministration shown. (c) C57BL/6 mice (n = 3) received a single intravenous dose of 3 mg/kg of Factor VII targeting siRNA formulated in lipidoid nanoparticles with different mean particle sizes. Control animals received saline injections. Serum Factor VII protein levels were measured 48 hours postadministration. Data points represent group mean ± SD. PBS, phosphate-buffered saline; PEG, poly(ethylene glycol); siRNA, small interfering RNA.

Figure 4

Key physical attributes of LNP01. (a) Volume-averaged particle size distribution by dynamic light scattering. Data shown as an overlay of three independent measurements. (b) Zeta potential measurement in 0.1× PBS. Data shown as an overlay of three independent measurements. (c) Serum stability of unformulated and LNP01-formulated siRNA. Samples were incubated in human serum at 37 °C for various times, and integrity of siRNA was measured using high-performance liquid chromatography. PBS, phosphate-buffered saline; siRNA, small interfering RNA.

Figure 5

Activity is maintained upon repeat administration of LNP01. C57BL/6 mice (n = 5) received a bolus intravenous (IV) injection of either saline, LNP01-formulated control siRNA (siCont) at 5 mg/kg, or LNP01-formulated Factor VII targeting siRNA (siFVII) at 5 mg/kg. Serum samples were collected at various time points and Factor VII protein levels were determined in the samples. After recovery of Factor VII levels to baseline values, animals were redosed on the indicated days. Data were collected for three treatment cycles. Data are displayed as relative to saline-treated control values for each time point and represent mean values ± SD. siRNA, small interfering RNA.