Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes

Abstract

Single-stranded antisense oligonucleotides (SSOs) are used to modulate the expression of genes in animal models and are being investigated as potential therapeutics. To better understand why synthetic SSOs accumulate in the same intracellular location as the target RNA, we have isolated a novel mouse hepatocellular SV40 large T-antigen carcinoma cell line, MHT that maintains the ability to efficiently take up SSOs over several years in culture. Sequence-specific antisense effects are demonstrated at low nanomolar concentrations. SSO accumulation into cells is both time and concentration dependent. At least two distinct cellular pathways are responsible for SSO accumulation in cells: a non-productive pathway resulting in accumulation in lysosomes, and a functional uptake pathway in which the SSO gains access to the targeted RNA. We demonstrate that functional uptake, as defined by a sequence-specific reduction in target mRNA, is inhibited by brefeldin A and chloroquine. Functional uptake is blocked by siRNA inhibitors of the adaptor protein AP2M1, but not by clathrin or caveolin. Furthermore, we document that treatment of mice with an AP2M1 siRNA blocks functional uptake into liver tissue. Functional uptake of SSO appears to be mediated by a novel clathrin- and caveolin-independent endocytotic process.

Figure 1.

SSOs decrease SR-B1, PTEN and Malat1 expression in MHT cells and mouse liver. (A) MHT cells were transfected with SSOs using Lipofectin reagent for 4 h. Approximately 20 h after transfection, cells were harvested and the respective mRNA measured by real time RT–PCR. (B) SSOs were added to the growth medium, in the absence of cationic lipid for 48 h for free uptake. (C) Mice were given a single dose of SSOs (50, 16, 5 and 1.6 mg/kg). Seventy-two hours after dosing, animals were sacrificed and livers removed. Total RNA was extracted from the liver. mRNA levels for the respective genes was quantified using real-time RT–PCR. Mean values ± SDs (n = 5).

Figure 2.

Characterization of free uptake in MHT Cells. (A) The kinetics of SR-B1 SSO effects were determined by incubating cells in the presence of 1 µM SR-B1 SSO for the indicated times. (B) Target reduction and SSO uptake were compared using 10 µM fluorescein-conjugated SSO for the indicated times. Cell accumulation of SSO was determined by flow cytometry using the fluorescein labeled SSO. (C) Dose-dependent effects of SSO. The effects of the SR-B1 SSO on SR-B1 expression was determined by incubating MHT cells with increasing concentration of SSO for 24 h. The number of SSO molecules per cell was determined by mass spectrometry. (D) Kinetics of SSO association with MHT cells. MHT cells were incubated with increasing concentrations of SSOs for 15 min to 2 h, after which the cells were washed and incubated an additional 24 h before measuring target reduction. Mean values and SD were measured from triplicate samples.

Figure 3.

Functional uptake in MHT Cells is saturable. (A) MHT cells were incubated with increasing concentrations of SR-B1 SSO alone or the indicated concentration of SR-B1 SSO and increasing concentration of control (competitor) SSO. Twenty-four hours after adding the SSO to the cells, reduction in SR-B1 mRNA was measured by qRT–PCR. (B) DNA containing competitor SSOs are effecting functional uptake of SR-B1 SSO. MHT cells were incubated with 1 µM SR-B1 SSO in the presence of 10 µM competitor SSOs with increasing gap length. The more DNA present in the gap, the better the competition. (C) MHT cells and primary hepatocytes (D) were incubated with 100 nM SR-B1 SSO alone or in the presence of 5 µM competitor SSO of different lengths. The competitor SSOs had 2 MOE nucleotides on the 5′- and 3′-ends to help protect against nuclease degradation. The length of the DNA in the gap was varied as indicated. Twenty-four hours after adding the SSOs to the cells, SR-B1 mRNA was measured with qRT–PCR. SSOs with more DNA content were found to be better competitors. (E) Effect of different length competitors on functional uptake of SR-B1 SSO. MHT cells were incubated with 1 µM of SR-B1 SSO in the presence of 1 µM competitor SSOs. The length of the SSOs was varied by increasing the MOE nucleotides on the 5′- and 3′ wings with the number of DNA nucleotides in the center gap held constant at 10 nt. Mean values and SD were measured from triplicate samples, *P < 0.01, **P < 0.05, unpaired Student’s t-test.

Figure 4.

Dextran sulfate competes for bulk uptake and functional SSO uptake. Increasing doses of SR-B1 SSO was added to complete medium with or without the indicated concentrations of dextran sulfate for 24 h. (A) High concentrations (10 µM) dextran sulfate decrease the pharmacological effects of the SR-B1 SSO while 1 µM dextran sulfate does not inhibit the pharmacological effects of the SR-B1 SSO. (B) Dextran sulfate inhibits SSO accumulation in MHT cells. MHT cells were incubated with 50 nM of fluorescein labeled SR-B1 SSO in the presence of increasing concentrations of dextrans sulfate for 24 h. At the end of the incubation period cells were collected and the amount of fluorescein labeled SSO determined by flow cytometry. Mean values and SD were determined from triplicate samples.

Figure 5.

Localization of fluorescently labeled SSO in MHT cells. (A) Fluorescein labeled SSO was transfected into MHT cells using lipofectin reagent for 4 h followed by 20 h of incubation in the absence of the cationic lipid in complete medium (left panel) or delivered to cells by adding to complete medium for 24 h (right panel). SSO localization is shown in formaldehyde fixed cells. (B) MHT cells were incubated with 100 nM fluorescein labeled SR-B1 SSO (green) for 2 h and cells were fixed and permeabilized. Cells were stained with a LAMP1 antibody (red) to label lysosomal structures and visualized using confocal microscopy. Nuclei of the cells were counterstained with DAPI. (C) MHT cells were incubated with 100 nM fluorescein labeled SR-B1 SSO (green) for 24 h and cells were fixed and permeabilized. Cells were stained with a LAMP1 antibody (red) to label lysosomal structures and visualized using confocal microscopy. Nuclei of the cells were counterstained with DAPI (blue). (D) MHT cells were incubated with 100 nM unlabeled SR-B1 SSO (top row), fluorescein conjugated SR-B1 SSO or Cy3 labeled SR-B1 SSO for 24 h. At the end of the incubation period, cells were fixed and stained for LAMP1 using an unlabeled LAMP1 monoclonal antibody, followed by either a fluorescein (top and bottom rows) or Texas red (middle row) labeled goat anti-mouse antibody. Cells were counterstained with DAPI (blue dye). The unlabeled antibody, was detected using a rabbit polyclonal antibody that recognizes the phosphorothioate backbone present in the SSO followed by a Texas red goat anti-rabbit antibody.

Figure 6.

SSO mediated functional uptake in MHT cells is AP2M1 dependent and clathrin independent. (A) MHT cells were treated with 25 nM control, AP2M1, clathrin or RNAse H1 siRNAs 2 days prior to adding 80 nM SR-B1 SSO. Cells were incubated an additional 24 h with the SR-B1 SSO and SR-B1 mRNA levels determined by qRT–PCR. SR-B1 mRNA reduction in AP2M1 siRNA-treated cells are inhibited compared to control siRNA-treated cells. (B and C) MHT cells were treated with 25 nM control siRNA, AP2M1 siRNA or no siRNA. Forty-eight hours later, increasing concentrations of the SR-B1 SSO were added to the cells in complete medium for an additional 24 h. Total cell RNA was isolated and SR-B1 (B) and AP2M1 (C) mRNAs were measured using qRT-PCR. (D) MHT cells were treated with 25 nM clathrin, AP2M1 or control siRNAs for 48 h, after which the cells were incubated with 100 nM fluorescently labeled transferrin-AF488 for 24 h. Uptake of Transferrin-AF488 was measured using flow cytometry. Cells were incubated with 10 µM chlorpromazine for 2 h prior to adding Transferrin-AF488 for 7 h. Mean values and SD were measured from triplicate samples, *P < 0.001, unpaired Student’s t-test.

Figure 7.

Inhibition of AP2M1 expression in mouse liver attenuates SR-B1 target reduction. (A–D) Balb/C mice were treated with 10 mg/kg nanoparticle formulated siRNAs on days 1 and 7 before dosing 20 mg/kg of the unformulated SR-B1 SSO on day 14. Mice were euthanized on day 17 (3 days after treating with SSO) and livers removed. (A) Total RNA was isolated and SR-B1 mRNA quantitated using qRT-PCR. Mean values ± SDs (n = 5). Reduction of SR-B1 levels in AP2M1 siRNA-treated mice are inhibited compared to luciferase siRNA-treated mice. (B) AP2M1 mRNA was determined in liver samples treated as indicated using qRT-PCR. (C) SR-B1 and (D) AP2M1 protein levels were determined using western blotting. Mean values ± SDs (n = 5), *P < 0.05, unpaired Student’s t-test.