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GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells

View Article: PubMed Central - PubMed

ABSTRACT

Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to “hard-to-transfect” primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called “GapmeR”, is an emerging new class of gene silencing molecule. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis. Internalized GapmeR molecules can associate with SNX5-positive macropinosomes in T-cells, as detected by super-resolution microscopy. Utilizing the intrinsic self-internalizing capability of GapmeR, we demonstrate significant and specific depletion (>70%) of the expression of 5 different endogenous proteins with varying molecular weights (18 kDa Stathmin, 80 kDa PKCε, 180 kDa CD11a, 220 kDa Talin1 and 450 kDa CG-NAP/AKAP450) in human primary and cultured T-cells. Further functional analysis confirms CG-NAP and Stathmin as regulators of T-cell motility. Thus, in addition to screening, identifying or verifying critical roles of various proteins in T-cell functioning, this study provides novel opportunities to silence individual or multiple genes in a subset of purified human primary T-cells that would be exploited as future therapeutics.

No MeSH data available.


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Functional effect of GapmeR-mediated gene silencing of CG-NAP, Talin1, CD11a, PKCε or Stathmin on T-cell migration.(A) Control or GapmeR treated primary human T-cells (2 × 104 cells in 100 μl medium) were loaded in triplicates onto rICAM-1-Fc pre-coated 96-well tissue culture plates and allowed to migrate at 37 °C for 4 h. Resting T-cells were incubated on poly L-lysine (PLL)-coated plates and cells pre-treated with nocodazole were used as migration inhibitory control. T-cell migratory phenotypes were then automatically quantified using HCA system (cell 1/form-factor) and presented as a heatmap. (B) Control or GapmeR treated primary human T-cells (1 × 105 cells in 100 μl medium) were loaded in triplicates onto the upper chamber of rICAM-1-Fc pre-coated CIM-Plate 16 transwell inserts. T-cell transwell migration towards SDF-1α enriched medium was automatically recorded in real-time at every 5 min interval for up to 6 h using an impedance-based detection system and quantified as “Baseline Cell Index”. Data represent at least three independent experiments using T-cells purified from at least 3 different donors.
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f6: Functional effect of GapmeR-mediated gene silencing of CG-NAP, Talin1, CD11a, PKCε or Stathmin on T-cell migration.(A) Control or GapmeR treated primary human T-cells (2 × 104 cells in 100 μl medium) were loaded in triplicates onto rICAM-1-Fc pre-coated 96-well tissue culture plates and allowed to migrate at 37 °C for 4 h. Resting T-cells were incubated on poly L-lysine (PLL)-coated plates and cells pre-treated with nocodazole were used as migration inhibitory control. T-cell migratory phenotypes were then automatically quantified using HCA system (cell 1/form-factor) and presented as a heatmap. (B) Control or GapmeR treated primary human T-cells (1 × 105 cells in 100 μl medium) were loaded in triplicates onto the upper chamber of rICAM-1-Fc pre-coated CIM-Plate 16 transwell inserts. T-cell transwell migration towards SDF-1α enriched medium was automatically recorded in real-time at every 5 min interval for up to 6 h using an impedance-based detection system and quantified as “Baseline Cell Index”. Data represent at least three independent experiments using T-cells purified from at least 3 different donors.

Mentions: Finally, to examine the functional influence of GapmeR-mediated gene silencing of the above 5 molecules in human primary T-cells, we performed our well-established T-cell migration assays24. Human primary T-cells depleted with the individual proteins (CG-NAP/AKAP9, Talin1, CD11a, PKCε or Stathmin using specific GapmeR molecules) were allowed to migrate through LFA-1/rICAM-1 cross-linking for 4 h as described previously24. High Content Analysis of the cells showed significant inhibition of T-cell migratory phenotypes due to the silencing of either of the above genes, including CG-NAP/AKAP9 and Stathmin (Fig. 6A). In addition, specific depletion of the selected 5 proteins individually inhibited T-cell transwell migration through ICAM-1-coated membranes towards the chemokine SDF1α, as analysed using a real-time impedance-based detection system (Fig. 6B).


GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells
Functional effect of GapmeR-mediated gene silencing of CG-NAP, Talin1, CD11a, PKCε or Stathmin on T-cell migration.(A) Control or GapmeR treated primary human T-cells (2 × 104 cells in 100 μl medium) were loaded in triplicates onto rICAM-1-Fc pre-coated 96-well tissue culture plates and allowed to migrate at 37 °C for 4 h. Resting T-cells were incubated on poly L-lysine (PLL)-coated plates and cells pre-treated with nocodazole were used as migration inhibitory control. T-cell migratory phenotypes were then automatically quantified using HCA system (cell 1/form-factor) and presented as a heatmap. (B) Control or GapmeR treated primary human T-cells (1 × 105 cells in 100 μl medium) were loaded in triplicates onto the upper chamber of rICAM-1-Fc pre-coated CIM-Plate 16 transwell inserts. T-cell transwell migration towards SDF-1α enriched medium was automatically recorded in real-time at every 5 min interval for up to 6 h using an impedance-based detection system and quantified as “Baseline Cell Index”. Data represent at least three independent experiments using T-cells purified from at least 3 different donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121623&req=5

f6: Functional effect of GapmeR-mediated gene silencing of CG-NAP, Talin1, CD11a, PKCε or Stathmin on T-cell migration.(A) Control or GapmeR treated primary human T-cells (2 × 104 cells in 100 μl medium) were loaded in triplicates onto rICAM-1-Fc pre-coated 96-well tissue culture plates and allowed to migrate at 37 °C for 4 h. Resting T-cells were incubated on poly L-lysine (PLL)-coated plates and cells pre-treated with nocodazole were used as migration inhibitory control. T-cell migratory phenotypes were then automatically quantified using HCA system (cell 1/form-factor) and presented as a heatmap. (B) Control or GapmeR treated primary human T-cells (1 × 105 cells in 100 μl medium) were loaded in triplicates onto the upper chamber of rICAM-1-Fc pre-coated CIM-Plate 16 transwell inserts. T-cell transwell migration towards SDF-1α enriched medium was automatically recorded in real-time at every 5 min interval for up to 6 h using an impedance-based detection system and quantified as “Baseline Cell Index”. Data represent at least three independent experiments using T-cells purified from at least 3 different donors.
Mentions: Finally, to examine the functional influence of GapmeR-mediated gene silencing of the above 5 molecules in human primary T-cells, we performed our well-established T-cell migration assays24. Human primary T-cells depleted with the individual proteins (CG-NAP/AKAP9, Talin1, CD11a, PKCε or Stathmin using specific GapmeR molecules) were allowed to migrate through LFA-1/rICAM-1 cross-linking for 4 h as described previously24. High Content Analysis of the cells showed significant inhibition of T-cell migratory phenotypes due to the silencing of either of the above genes, including CG-NAP/AKAP9 and Stathmin (Fig. 6A). In addition, specific depletion of the selected 5 proteins individually inhibited T-cell transwell migration through ICAM-1-coated membranes towards the chemokine SDF1α, as analysed using a real-time impedance-based detection system (Fig. 6B).

View Article: PubMed Central - PubMed

ABSTRACT

Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to “hard-to-transfect” primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called “GapmeR”, is an emerging new class of gene silencing molecule. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis. Internalized GapmeR molecules can associate with SNX5-positive macropinosomes in T-cells, as detected by super-resolution microscopy. Utilizing the intrinsic self-internalizing capability of GapmeR, we demonstrate significant and specific depletion (>70%) of the expression of 5 different endogenous proteins with varying molecular weights (18 kDa Stathmin, 80 kDa PKCε, 180 kDa CD11a, 220 kDa Talin1 and 450 kDa CG-NAP/AKAP450) in human primary and cultured T-cells. Further functional analysis confirms CG-NAP and Stathmin as regulators of T-cell motility. Thus, in addition to screening, identifying or verifying critical roles of various proteins in T-cell functioning, this study provides novel opportunities to silence individual or multiple genes in a subset of purified human primary T-cells that would be exploited as future therapeutics.

No MeSH data available.


Related in: MedlinePlus