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

Koller E, Vincent TM, Chappell A, De S, Manoharan M, Bennett CF - Nucleic Acids Res. (2011)

Bottom Line: Sequence-specific antisense effects are demonstrated at low nanomolar concentrations.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.

View Article: PubMed Central - PubMed

Affiliation: Isis Pharmaceuticals Inc., 1896 Rutherford Road, Carlsbad, CA 92008 and Alnylam Pharmaceuticals Inc., 300 Third Street, Cambridge, MA 02142, USA. ekoller@isisph.com

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.

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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.
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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.

Mentions: To further define the pathways involved we chose to inhibit the expression of specific genes using siRNA. We chose to use siRNA for these experiments as they exploit a different molecular mechanism than SSOs and therefore should not result in competition for RNase H or other components required for the SSOs used in these studies, potentially complicating interpretation of the data. We screened a mouse siRNA library targeting membrane trafficking proteins for effects on antisense activity of the SR-B1 SSO (35). As a positive control we demonstrated that RNAse H1 siRNAs (Figure 6A) decreased the activity of the SSOs, indicating that they work through an RNAse H1 dependent mechanism. In this experiment the RNase H siRNAs reduce the amount of RNase H mRNA by 75%. The strongest and most reproducible hit for inhibiting the effects of the SR-B1 SSO was the adaptor protein AP2M1. Inhibition of AP2M1 with either a pool of four siRNAs or individual siRNAs in the pool attenuated the antisense effects of SR-B1 SSO (Figure 6B). Treatment with the AP2M1 siRNA pool resulted in an 80% inhibition of AP2M1 mRNA levels (Figure 6C). Inhibition of AP2M1 expression decreased fluorescein labeled SSO uptake by 50% (Supplementary Figure S7A). AP2M1 is an adaptor protein that recognizes a peptide sequence on the cytoplasmic face of cell membrane proteins linking the cell membrane cargo to clathrin (36,37). Surprisingly, inhibition of clathrin expression using siRNAs (80% inhibition, data not shown) had no effect on the SSO activity (Figure 6A and Supplementary Figure 6B), but did decrease the uptake of fluorescently labeled transferrin uptake in MHT cells, as did the AP2M1 siRNA and chlorpromazine (Figure 6D). These results demonstrate that functional uptake of SSO into MHT cells is AP2M1-dependent, but clathrin independent. Inhibition of caveolin expression also had no effect on functional SSO uptake (data not shown).Figure 6.


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

Koller E, Vincent TM, Chappell A, De S, Manoharan M, Bennett CF - Nucleic Acids Res. (2011)

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.
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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.
Mentions: To further define the pathways involved we chose to inhibit the expression of specific genes using siRNA. We chose to use siRNA for these experiments as they exploit a different molecular mechanism than SSOs and therefore should not result in competition for RNase H or other components required for the SSOs used in these studies, potentially complicating interpretation of the data. We screened a mouse siRNA library targeting membrane trafficking proteins for effects on antisense activity of the SR-B1 SSO (35). As a positive control we demonstrated that RNAse H1 siRNAs (Figure 6A) decreased the activity of the SSOs, indicating that they work through an RNAse H1 dependent mechanism. In this experiment the RNase H siRNAs reduce the amount of RNase H mRNA by 75%. The strongest and most reproducible hit for inhibiting the effects of the SR-B1 SSO was the adaptor protein AP2M1. Inhibition of AP2M1 with either a pool of four siRNAs or individual siRNAs in the pool attenuated the antisense effects of SR-B1 SSO (Figure 6B). Treatment with the AP2M1 siRNA pool resulted in an 80% inhibition of AP2M1 mRNA levels (Figure 6C). Inhibition of AP2M1 expression decreased fluorescein labeled SSO uptake by 50% (Supplementary Figure S7A). AP2M1 is an adaptor protein that recognizes a peptide sequence on the cytoplasmic face of cell membrane proteins linking the cell membrane cargo to clathrin (36,37). Surprisingly, inhibition of clathrin expression using siRNAs (80% inhibition, data not shown) had no effect on the SSO activity (Figure 6A and Supplementary Figure 6B), but did decrease the uptake of fluorescently labeled transferrin uptake in MHT cells, as did the AP2M1 siRNA and chlorpromazine (Figure 6D). These results demonstrate that functional uptake of SSO into MHT cells is AP2M1-dependent, but clathrin independent. Inhibition of caveolin expression also had no effect on functional SSO uptake (data not shown).Figure 6.

Bottom Line: Sequence-specific antisense effects are demonstrated at low nanomolar concentrations.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.

View Article: PubMed Central - PubMed

Affiliation: Isis Pharmaceuticals Inc., 1896 Rutherford Road, Carlsbad, CA 92008 and Alnylam Pharmaceuticals Inc., 300 Third Street, Cambridge, MA 02142, USA. ekoller@isisph.com

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.

Show MeSH
Related in: MedlinePlus