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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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Related in: MedlinePlus

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.
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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.

Mentions: The localization of a fluorescein-labeled SSO was determined in the presence or absence of cationic lipid. Similar to previous results (15), we found that the SSO primarly localized in the nucleus when delivered into the cells with cationic lipids (Figure 5A). In the absence of cationic lipid, the highest concentration of SSO was found in punctuate peri-nuclear structures (Figure 5A–D). The same SSO distribution was seen in fixed and live cells (data not shown). We used antibodies to the lysosomal membrane protein 1 (LAMP1) to determine if the SSO co-localized in lysosomal structures. Two hours after adding the fluorescein-conjugated SSO to cells, SSOs localized to punctuate structures in the cytoplasm but did not co-localize with lysosomes (Figure 5B). However, after 24 h most of the fluorescent SSO co-localized with lysosomes (Figure 5C), suggesting that SSO accumulation in lysosomes is a slow process in these cells. Unconjugated SSO also localized in punctuate peri-nuclear structures as demonstrated using a polyclonal antibody that recognizes phosphorothioate modified SSO. The localization of the unlabeled SSO was similar to SSO labeled with fluorescein or Cy3 (Figure 5D).Figure 5.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: The localization of a fluorescein-labeled SSO was determined in the presence or absence of cationic lipid. Similar to previous results (15), we found that the SSO primarly localized in the nucleus when delivered into the cells with cationic lipids (Figure 5A). In the absence of cationic lipid, the highest concentration of SSO was found in punctuate peri-nuclear structures (Figure 5A–D). The same SSO distribution was seen in fixed and live cells (data not shown). We used antibodies to the lysosomal membrane protein 1 (LAMP1) to determine if the SSO co-localized in lysosomal structures. Two hours after adding the fluorescein-conjugated SSO to cells, SSOs localized to punctuate structures in the cytoplasm but did not co-localize with lysosomes (Figure 5B). However, after 24 h most of the fluorescent SSO co-localized with lysosomes (Figure 5C), suggesting that SSO accumulation in lysosomes is a slow process in these cells. Unconjugated SSO also localized in punctuate peri-nuclear structures as demonstrated using a polyclonal antibody that recognizes phosphorothioate modified SSO. The localization of the unlabeled SSO was similar to SSO labeled with fluorescein or Cy3 (Figure 5D).Figure 5.

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