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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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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.
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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.

Mentions: We extended these observations to mouse liver. AP2M1 or luciferase siRNAs were administered to mice on day 1 and 7 using a lipidoid nanoparticle (LNP) formulation (38). An unformulated SSO targeting SR-B1 was injected subcutaneously on day 14 and mice were sacrificed on day 17. As observed in MHT cells, inhibition of AP2M1 in mouse liver blunted the antisense effects of the SR-BI SSO while the luciferase siRNA had no effect on SR-B1 reduction (Figure 7A). The AP2M1 siRNA resulted in 80% reduction of mouse AP2M1 mRNA (Figure 7B). Inhibition of AP2M1 also inhibited SR-B1 protein reduction by the SR-B1 SSO (Figure 7C). The siRNA reduced AP2M1 protein by 60% (Figure 7D).Figure 7.


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)

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.
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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.
Mentions: We extended these observations to mouse liver. AP2M1 or luciferase siRNAs were administered to mice on day 1 and 7 using a lipidoid nanoparticle (LNP) formulation (38). An unformulated SSO targeting SR-B1 was injected subcutaneously on day 14 and mice were sacrificed on day 17. As observed in MHT cells, inhibition of AP2M1 in mouse liver blunted the antisense effects of the SR-BI SSO while the luciferase siRNA had no effect on SR-B1 reduction (Figure 7A). The AP2M1 siRNA resulted in 80% reduction of mouse AP2M1 mRNA (Figure 7B). Inhibition of AP2M1 also inhibited SR-B1 protein reduction by the SR-B1 SSO (Figure 7C). The siRNA reduced AP2M1 protein by 60% (Figure 7D).Figure 7.

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