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WIREs Nanomed Nanobiotechnol
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T‐cell targeted pulmonary siRNA delivery for the treatment of asthma

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Abstract Despite the large number of drugs available for the treatment of asthma, in 5–10% of the patients this disease is not well controlled. While most treatments palliate symptoms, those suffering from severe and uncontrolled asthma could benefit more from a therapeutic approach addressing the root problem. An siRNA‐based therapy targeting the transcription factor GATA3 in activated T helper cells subtype 2 (TH2 cells), one of the key upstream factors involved in asthma, could therefore represent a promising strategy. However, the difficult‐to‐transfect cell type has not extensively been explored for nucleic acid therapeutics. In this regard, our group first identified a suitable pathway, that is, transferrin receptor mediated uptake, to target efficiently and specifically activated TH2 cells with a transferrin‐polyethyleneimine (PEI) conjugate which forms polyplexes with siRNA. This system, despite efficient uptake in activated T cells (ATCs) in vivo, suffered from poor endosomal release and was later improved by a combination with a melittin‐PEI conjugate. The new formulation showed improved endosomal escape and gene silencing efficacy. Additionally, in order to develop a clinically relevant dosage form for pulmonary delivery of siRNA we have lately focused on a dry powder formulation by spray drying (SD) for the production of inhalable nano‐in‐microparticles. In proof‐of‐concept experiments, DNA/PEI polyplexes were used in order to implement analytics and engineer process parameters to pave the way for SD also siRNA containing polyplexes and more sophisticated systems in general. Ultimately, our efforts are devoted to the development of a novel treatment of asthma that can be translated from bench to bedside and are reviewed and discussed here in the context of the current literature. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Respiratory Disease Biology‐Inspired Nanomaterials > Nucleic Acid‐Based Structures Biology‐Inspired Nanomaterials > Protein and Virus‐Based Structures
T helper‐2 (TH2) cells in asthma pathogenesis. Inhaled allergens are thought to be processed by two mechanisms in asthmatic airways. Allergens either: (1) activate mast cells through cross‐linking with IgE on their cell surfaces through the high‐affinity type 1 IgE receptor (FcɛR1) to release mediators that induce bronchoconstriction, such as histamine, cysteinyl leukotrienes, and prostaglandin D2 (PGD2) or (2) are processed by dendritic cells, which are induced to secrete the CC chemokine ligand (CCL) 17 and CCL22 by thymic stromal lymphopoietin (TSLP). Dendritic cells then attract and activate TH2 cells by the binding of CCL17 and CCL22 with CC chemokine receptor 4 (CCR4) on the TH2 cell surface. IL‐33 is produced by airway epithelial cells and activates dendritic cells and TH2 by inducing the release of TNF‐α from mast cells. TH2 secretes cytokines, including IL‐4 and IL‐13, which switch B cells to produce IgE, IL‐5, which promotes the development and survival of eosinophils, and IL‐9, which activates mast cells. Once IL‐13 is produced, it can increase the survival and migration of eosinophils, and it promotes activation of macrophages to create an M2, or an allergic cell phenotype. Airway epithelial cells are stimulated, and through mediators such as periostin and transforming growth factor β1 (TGF‐β1), they can increase airway inflammation and lead to the increased permeability of airway epithelial cells and mucous hypersecretion. IL‐13 also has direct effects on airway smooth muscle, leading to increased contraction to agonists such as acetylcholine and decreased relaxation with beta‐agonists. (Reproduced with permission from Thomson, Patel, and Smith (2012). Copyright 2012 Dove Medical Press Ltd.)
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Polymer and nucleic acid quantification. Quantification of (a) bulk DNA and (b) PEI in the 10% trehalose nano‐in‐microparticle (NIM) formulations following spray drying; (c) comparing the redispersed N/P ratio with the initial N/P ratio of the PEI polyplexes loaded with bulk DNA; (d) uptake and (e) transfection efficiency of redispersed NIM formulations in A549 cells. Median fluorescence intensity (MFI) was determined by flow cytometry to evaluate efficiency of (d) fluorescently labeled bDNA or E) pEGFP in a human nonsmall cell lung carcinoma cell line (A549) of fresh or redispersed polyplexes from 10% trehalose NIM formulations at N/P ratios of 6, 8 and 10 with (d) 0.5 μg of bDNA or E) 0.75 μg of GFP plasmid in comparison to freshly prepared formulations in presence of trehalose. Blank samples consisted of A549 cells treated with 5% glucose only. Data points indicate mean ± SD (n = 3). Two‐way analysis of variance, Bonferroni post‐test, *p < .05, **p < .01, *** p < .001, ns, nonsignificant. (Reproduced with permission from Keil et al. (2019). Copyright 2019 Elsevier B.V.)
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(a) Acridine orange staining of untreated A549 cells (A1) and of A549 cells after incubation of polyplexes with Chloroquine (A2), PEI (A3), Tf‐PEI (A4), Mel‐PEI (A5) and Tf‐Mel‐PEI (A6). (b) GAPDH knockdown in Jurkat cells (B1) and human primary activated T cells (B2) after treatment with GAPDH‐siRNA or scrambled siRNA as negative control. Data points indicate mean ± SD, n = 3; One‐way analysis of variance, *p < .05, **p < .01, *** p < .001. (Reproduced with permission from Kandil, Feldmann, et al. (2019). Copyright 2019 Wiley)
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Specific Uptake of siRNA in activated T cells with a Tf‐PEI conjugate. Uptake of Alexa488‐labaled siRNA (a) at different N/P ratios into fully activated T cells with high TfR expression (inset 2a), and (b) lack of uptake into T cells with low TfR expression (inset 2b). The expression of TfR in the T cells was confirmed by anti‐CD71 antibody binding assay. The siRNA taken up into T cells was analyzed by flow cytometry. Lipofectamine was used as a positive control. (Reproduced with permission from Kim et al. (2013). Copyright 2013 Elsevier B.V.)
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Biology-Inspired Nanomaterials > Protein and Virus-Based Structures
Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures
Therapeutic Approaches and Drug Discovery > Nanomedicine for Respiratory Disease

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