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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.


Effect of GapmeR treatment on T-cell viability and immunogenic responses.(A) Human primary T-cells were incubated with 1 μM non-targeting GapmeR to allow gymnosis or transfected by nucleofection. Equivalent amount of PBS was used as control. After 48 h, percentage cell viability was determined and presented. (B–E) Human primary T-cells were incubated with 100, 250 or 500 nM non-targeting GapmeR for 24 h. Cells were left untreated (NT) or treated with phytohemagglutinin (PHA) as negative and positive controls. After centrifugation, supernatant culture media were collected and secreted levels of cytokines IL-2 (B), IL-4 (C), IL-5 (D) and IFN-γ (E) were analysed by ELISA. Data (mean ± SEM) represent three independent experiments using T-cells purified from at least 3 different donors, *p < 0.05.
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f2: Effect of GapmeR treatment on T-cell viability and immunogenic responses.(A) Human primary T-cells were incubated with 1 μM non-targeting GapmeR to allow gymnosis or transfected by nucleofection. Equivalent amount of PBS was used as control. After 48 h, percentage cell viability was determined and presented. (B–E) Human primary T-cells were incubated with 100, 250 or 500 nM non-targeting GapmeR for 24 h. Cells were left untreated (NT) or treated with phytohemagglutinin (PHA) as negative and positive controls. After centrifugation, supernatant culture media were collected and secreted levels of cytokines IL-2 (B), IL-4 (C), IL-5 (D) and IFN-γ (E) were analysed by ELISA. Data (mean ± SEM) represent three independent experiments using T-cells purified from at least 3 different donors, *p < 0.05.

Mentions: There was no appreciable loss in cell viability due to the incubation of T-cells with as high as 1 μM non-targeting GapmeR for 24 h (Fig. 2A, green bars); however, nucleofection procedure alone caused significant loss in cell viability (Fig. 2A, red bars). Moreover, GapmeR treatment did not induce any unwanted immunogenic response in human primary T-cells i.e. there was no detectable secretion of IL-2, IL-4, IL-5 or IFN-γ due to non-targeting GapmeR (Fig. 2B–E).


GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells
Effect of GapmeR treatment on T-cell viability and immunogenic responses.(A) Human primary T-cells were incubated with 1 μM non-targeting GapmeR to allow gymnosis or transfected by nucleofection. Equivalent amount of PBS was used as control. After 48 h, percentage cell viability was determined and presented. (B–E) Human primary T-cells were incubated with 100, 250 or 500 nM non-targeting GapmeR for 24 h. Cells were left untreated (NT) or treated with phytohemagglutinin (PHA) as negative and positive controls. After centrifugation, supernatant culture media were collected and secreted levels of cytokines IL-2 (B), IL-4 (C), IL-5 (D) and IFN-γ (E) were analysed by ELISA. Data (mean ± SEM) represent three independent experiments using T-cells purified from at least 3 different donors, *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121623&req=5

f2: Effect of GapmeR treatment on T-cell viability and immunogenic responses.(A) Human primary T-cells were incubated with 1 μM non-targeting GapmeR to allow gymnosis or transfected by nucleofection. Equivalent amount of PBS was used as control. After 48 h, percentage cell viability was determined and presented. (B–E) Human primary T-cells were incubated with 100, 250 or 500 nM non-targeting GapmeR for 24 h. Cells were left untreated (NT) or treated with phytohemagglutinin (PHA) as negative and positive controls. After centrifugation, supernatant culture media were collected and secreted levels of cytokines IL-2 (B), IL-4 (C), IL-5 (D) and IFN-γ (E) were analysed by ELISA. Data (mean ± SEM) represent three independent experiments using T-cells purified from at least 3 different donors, *p < 0.05.
Mentions: There was no appreciable loss in cell viability due to the incubation of T-cells with as high as 1 μM non-targeting GapmeR for 24 h (Fig. 2A, green bars); however, nucleofection procedure alone caused significant loss in cell viability (Fig. 2A, red bars). Moreover, GapmeR treatment did not induce any unwanted immunogenic response in human primary T-cells i.e. there was no detectable secretion of IL-2, IL-4, IL-5 or IFN-γ due to non-targeting GapmeR (Fig. 2B–E).

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 &ldquo;hard-to-transfect&rdquo; primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called &ldquo;GapmeR&rdquo;, 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 (&gt;70%) of the expression of 5 different endogenous proteins with varying molecular weights (18&thinsp;kDa Stathmin, 80&thinsp;kDa PKC&epsilon;, 180&thinsp;kDa CD11a, 220&thinsp;kDa Talin1 and 450&thinsp;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.