Correction: RNA interference-mediated gene silencing in murine T cells: in vitro and in vivo validation of proinflammatory target genes

Gust, Tatjana C; Luisa, Neubrandt; Merz, Claudia; Asadullah, Khusru; Zügel, Ulrich; Bonin, Arne von

doi:10.1186/1478-811x-6-3

Cited by 13 publications

(9 citation statements)

References 24 publications

(22 reference statements)

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“…As a third approach to investigate the role of Itk in T‐cell function, RNA interference (RNAi) experiments were performed (38–40). To test whether Itk knockdown in human T cells leads to impaired T‐cell activation comparable to the situation observed in Itk knockout mice and in mice treated with the Itk inhibitor, purified human T cells were transfected with Itk‐specific or control siRNA.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of the IL-2-inducible tyrosine kinase (Itk) activity: a new concept for the therapy of inflammatory skin diseases

Bonin

Rausch

Mengel

et al. 2010

Experimental Dermatology

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T-cell-mediated processes play an essential role in the pathogenesis of several inflammatory skin diseases such as atopic dermatitis, allergic contact dermatitis and psoriasis. The aim of this study was to investigate the role of the IL-2-inducible tyrosine kinase (Itk), an enzyme acting downstream of the T-cell receptor (TCR), in T-cell-dependent skin inflammation using three approaches. Itk knockout mice display significantly reduced inflammatory symptoms in mouse models of acute and subacute contact hypersensitivity (CHS) reactions. Systemic administration of a novel small molecule Itk inhibitor, Compound 44, created by chemical optimization of an initial high-throughput screening hit, inhibited Itk's activity with an IC50 in the nanomolar range. Compound 44 substantially reduced proinflammatory immune responses in vitro and in vivo after systemic administration in two acute CHS models. In addition, our data reveal that human Itk, comparable to its murine homologue, is expressed mainly in T cells and is increased in lesional skin from patients with atopic dermatitis and allergic contact dermatitis. Finally, silencing of Itk by RNA interference in primary human T cells efficiently blocks TCR-induced lymphokine secretion. In conclusion, Itk represents an interesting new target for the therapy of T-cell-mediated inflammatory skin diseases.

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Section: Resultsmentioning

confidence: 99%

Inhibition of the IL-2-inducible tyrosine kinase (Itk) activity: a new concept for the therapy of inflammatory skin diseases

Bonin

Rausch

Mengel

et al. 2010

Experimental Dermatology

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“…Gene silencing appears to be more efficient if T-cells are activated through the TCR either alone or in combination with CD28 before electroporation/nucleofection [37,42,49,50], which correlates with studies that have reported higher levels of gene expression in activated primary T-cells using plasmids compared with nonactivated T-cells [28,30,32]. Gene silencing appears to be more efficient if T-cells are activated through the TCR either alone or in combination with CD28 before electroporation/nucleofection [37,42,49,50], which correlates with studies that have reported higher levels of gene expression in activated primary T-cells using plasmids compared with nonactivated T-cells [28,30,32].…”

Section: Mechanismmentioning

confidence: 99%

“…For example, knockdown of the tyrosine kinase ZAP-70 in murine T-cells via nucleofection with siRNAs, followed by adoptive transfer of these cells into recipient mice suppresses delayed type hypersensitivity (a Th1-mediated inflammatory response) [49]. For example, knockdown of the tyrosine kinase ZAP-70 in murine T-cells via nucleofection with siRNAs, followed by adoptive transfer of these cells into recipient mice suppresses delayed type hypersensitivity (a Th1-mediated inflammatory response) [49].…”

Section: Therapeutic Opportunitiesmentioning

confidence: 99%

“…In relation to this point, Ward and colleagues reported that nucleofection of naïve primary human T-cells with siRNAs followed by cellular stimulation resulted in high levels of cell death [54]. Nucleofection and electroporation of primary T-cells also generally require >10-fold higher concentrations of siRNA for effective gene silencing compared with lipid-based silencing protocols for adherent cells [37,41,49,51,53,54], which in addition to the nucleofection reagents makes this delivery method quite expensive when performed on a routine basis or when performing siRNA screens [56]. In terms of the nucleofection system, both the nucleofection solutions and pre-set delivery programmes are proprietary knowledge, which limits the users' ability to develop alternative delivery conditions.…”

Section: Challengesmentioning

confidence: 99%

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Advances in siRNA delivery to T-cells: potential clinical applications for inflammatory disease, cancer and infection

Freeley

Long

2013

Biochemical Journal

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The specificity of RNAi and its ability to silence 'undruggable' targets has made inhibition of gene expression in T-cells with siRNAs an attractive potential therapeutic strategy for the treatment of inflammatory disease, cancer and infection. However, delivery of siRNAs into primary T-cells represents a major hurdle to their use as potential therapeutic agents. Recent advances in siRNA delivery through the use of electroporation/nucleofection, viral vectors, peptides/proteins, nanoparticles, aptamers and other agents have now enabled efficient gene silencing in primary T-cells both in vitro and in vivo. Overcoming such barriers in siRNA delivery offers exciting new prospects for directly targeting T-cells systemically with siRNAs, or adoptively transferring T-cells back into patients following ex vivo manipulation with siRNAs. In the present review, we outline the challenges in delivering siRNAs into primary T-cells and discuss the mechanism and therapeutic opportunities of each delivery method. We emphasize studies that have exploited RNAi-mediated gene silencing in T-cells for the treatment of inflammatory disease, cancer and infection using mouse models. We also discuss the potential therapeutic benefits of manipulating T-cells using siRNAs for the treatment of human diseases.

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“…For example, flow cytometric analysis should be run using the pancreata of 25-30 week-old wild type mice to confirm the expression of the desired proteins on a cell's surface [28,29], as RNA quantities obtained from microarray data do not always correlate with protein expression and occasionally yield false positives. Celltype-specific gene knock-out experiments can also be performed, either through in-vivo mouse models or in-vitro methods including standard siRNA procedures [30,31]. The use of these procedures can directly confirm the significance of the chosen genes in pancreatic Treg function and survival [32,33].…”

Section: Future Investigationsmentioning

confidence: 99%

Identification of Differentially Expressed Genes in Pancreatic Regulatory T-Cell Survivalc

Carrion

Carrion²

2015

J Diabetes Metab

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Objective: Many current Diabetes treatments cause broad-based immunodeficiency, though future organspecific therapies may be developed by exploiting genotypic differences found in regulatory T cell (Treg) subpopulations. This study goaled to determine genes preferentially expressed in pancreatic Tregs relative to other Treg subpopulations, tissues, and immune cells to serve as targets in Diabetes therapy.Methods: Multi-step microarray analysis using GenePattern Software identified genes specific to pancreatic Tregs relative to Tregs in other tissues and to other immune-cell populations. Functional analysis of identified genes was performed using BioGrid and String Databases. Microarray datasets (n=2236) curated from the NCBI GEO database were processed (via RMA normalization, outlier-array exclusion, and global median transformation) to confirm the specificity of these genes to the pancreas, while further microarray comparisons (in R programming language via Mas-5 normalization, log2 transformation, and "trimmed mean" algorithm) were performed to confirm the link of the identified genes to diabetes pathogenesis. Results:Initial microarray analysis identified genes specific to pancreatic Tregs, with the top three genes (Clps, Pnliprp1, and Pla2g1b) expressed at values 23x, 12x, and 7x higher in pancreatic Tregs than in other Treg subpopulations (p<0.05). Further analysis revealed that the pancreatic gene expression of the identified genes was almost triple that of other tissues (n=29), while comparisons among other immune-cell types indicated that these genes were expressed 32x, 12.8x, and 7.5x higher, respectively, in pancreatic Tregs than in other immune cell-types, eliminating the possibility of augmenting the autoimmune response in future treatments. Diabetic vs. nondiabetic microarray analysis confirmed the link of these genes to Type 1, but not Type 2, Diabetes.Conclusions: Future Diabetes therapies should target these genes to solely regulate pancreatic Treg levels, avoiding the broad-based immunosuppression caused by current therapies. However, gene knockout studies should be performed to further validate the functionality of the identified genes.

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Correction: RNA interference-mediated gene silencing in murine T cells: in vitro and in vivo validation of proinflammatory target genes

Cited by 13 publications

References 24 publications

Inhibition of the IL-2-inducible tyrosine kinase (Itk) activity: a new concept for the therapy of inflammatory skin diseases

Inhibition of the IL-2-inducible tyrosine kinase (Itk) activity: a new concept for the therapy of inflammatory skin diseases

Advances in siRNA delivery to T-cells: potential clinical applications for inflammatory disease, cancer and infection

Identification of Differentially Expressed Genes in Pancreatic Regulatory T-Cell Survivalc

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