Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms

Abstract

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

Figure 1

iLNPs but not cLNPs are dependent on apoE for cellular uptake and silencing in vitro. (a) Alexa-Fluor 647–labeled siRNA formulated in iLNPs or cLNPs were added to HeLa cells at 20 nmol/l and incubated for 4 hours at 37 °C under different media conditions: serum-free media, media with 10% FBS, or full media supplemented with 1 µg/ml of apoE. Cells were washed, fixed, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) then viewed by automated confocal microscopy. (b) Quantitation of cellular uptake. (c) Enhancement in cellular uptake achieved by preassociating increasing amounts of apoE with iLNPs or cLNPs. Data are expressed as fold increase over LNP without apoE. (d) cLNPs or iLNPs formulated with an Alexa-Fluor 647–labeled siRNA were incubated with primary hepatoctyes in the absence or presence of apoE for 4 hours. Cells were washed, fixed, and counterstained with DAPI, then viewed by automated confocal microscopy. (e) Silencing of GFP in HeLa-GFP cells following treatment of cells with iLNP and cLNP in the absence or presence of apoE. ApoE, apolipoprotein E; cLNP, cationic LNP; iLNP, ionizable LNP; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; LNP, lipid nanoparticle; RLU, relative light units; siRNA, short-interfering RNA.

Figure 2

iLNP activity in vivo is dependent on apoE. (a) In vivo factor VII gene-silencing activity of iLNPs and cLNPs in wild-type and apoE^−/− mice. FVII siRNA formulated in iLNPs or cLNPs were administered via bolus tail-vein injection to wild-type or apoE^−/− mice. Animals were killed at 48 hours postadministration and serum and livers were collected. Serum factor VII protein and liver factor VII mRNA levels were determined. Data points are expressed as a percentage of PBS control animals and represent group mean (n = 5) ± SD. (b) Rescue of iLNP activity in apoE^−/− mice using exogenous apoE. iLNPs containing FVII siRNA were premixed with increased amounts of recombinant human apoE protein and incubated overnight at 4 °C. The next day, iLNPs were administered to apoE^−/− mice at a constant siRNA dose of 0.2 mg/kg. Control animals received PBS or iLNPs without apoE protein. For comparison, wild-type mice received either PBS, iLNP, or iLNP associated with the highest concentration of apoE protein. Data points are expressed as a percentage of PBS control animals and represent group mean (n = 5) ± SD. ApoE, apolipoprotein E; cLNP, cationic LNP; FVII, factor VII; iLNP, ionizable LNP; LNP, lipid nanoparticle; PBS, phosphate-buffered saline; siRNA, short-interfering RNA; WT, wild type.

Figure 3

Loss of LDLR impairs iLNP activity in vitro and in vivo. (a) Primary hepatocytes were isolated from wild-type or LDLR^−/− mice. Cells were incubated with Alexa-Fluor 647–labeled siRNA formulated in iLNPs in the presence of apoE for 4 hours. Arrows indicate iLNPs containing labeled siRNA which appear to accumulate at the plasma membrane and fail to enter cells efficiently in LDLR^−/− hepatocytes. (b) Factor VII gene-silencing activity of iLNPs in wild-type and LDLR^−/− mice. FVII siRNA formulated in iLNPs were administered via bolus tail-vein injection. No exogenous apoE was added to the formulations. Animals were killed at 48 hours postadministration and serum samples were collected and analyzed for serum factor VII protein levels. Data points are expressed as a percentage of PBS control animals and represent group mean (n = 5) ± SD. FVII, factor VII; iLNP, ionizable LNP; LDLR, low-density lipoprotein receptor; LNP, lipid nanoparticle; PBS, phosphate-buffered saline; siRNA, short-interfering RNA; WT, wild type.

Figure 4

Receptor binding activity of GalNAc–iLNPs. (a) Structure of GalNAc–PEG–DSG. (b) Cell-free receptor competition-binding activity of iLNPs containing 0–0.5 mol% GalNAc–PEG–DSG. Data plotted as a function of siRNA concentration. (c) Competition-binding data replotted as a function of GalNAc concentration. DSG, distearyl (C18) lipid; GalNAc, N-acetylgalactosamine; iLNP, ionizable LNP; PEG, polyethylene glycol; siRNA, short-interfering RNA.

Figure 5

Exogenous targeting with GalNAc is able to rescue activity of iLNPs in apoE^−/− mice. (a) siFVII was formulated in GalNAc–iLNPs containing 0.005–0.5 mol% GalNAc–PEG–DSG and administered to apoE^−/− mice at a dose of 0.2 mg/kg. Control animals received PBS or FVII siRNA formulated in iLNP at 0.2 mg/kg. Serum factor VII levels were determined in animals 48 hours postadministration. Data points are expressed as a percentage of PBS control animals and represent group mean (n = 3) ± SD. (b) Comparative potency of 0.5% GalNAc–iLNP (containing 0.5 mol% of GalNAc–PEG–DSG) to iLNP preassociated with apoE (at a ratio of 0.5 mg apoE/mg siRNA) in apoE^−/− mice. Control animals received PBS or iLNP. Data points are expressed as a percentage of PBS control animals and represent group mean (n = 3) ± SD. (c) Factor VII gene-silencing activity of 0.5% GalNAc–iLNP in wild-type and LDLR^−/− mice. Serum factor VII levels were determined in animals 48 hours postadministration. Data points are expressed as a percentage of PBS control animals and represent group mean (n = 5) ± SD. ApoE, apolipoprotein E; DSG, distearyl (C18) lipid; GalNAc, N-acetylgalactosamine; iLNP, ionizable LNP; PBS, phosphate-buffered saline; PEG, polyethylene glycol; siFVII, factor VII–targeting siRNA; siRNA, short-interfering RNA.

Figure 6

GalNAc-targeted, “shielded” iLNPs demonstrate GalNAc-specific gene-silencing activity in vivo. FVII siRNA was formulated in a PEG-shielded GalNAc–iLNP containing ionizable lipid/DSPC/cholesterol/PEG–DSG/GalNAc–PEG–DSG in a 50/10/30/9.5/0.5 molar ratio. Wild-type or ASGR2^−/− mice were administered PBS, nontargeted shielded-iLNPs, or 0.5% GalNAc-shielded iLNPs at the doses indicated. Serum factor VII levels were determined in animals 48 hours postadministration. Data points are expressed as a percentage of PBS control animals and represent group mean (n = 5) ± SD. ASGR, asialoglycoprotein-binding receptor; DSG, distearyl (C18) lipid; GalNAc, N-acetylgalactosamine; iLNP, ionizable LNP; PBS, phosphate-buffered saline; PEG, polyethylene glycol; siFVII, factor VII–targeting siRNA; siRNA, short-interfering RNA.