A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5′ and 3′ end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy.

Air−liquid interface (ALI) culture models currently represent a valid instrument to recreate the typical aspects of the respiratory tract in vitro in both healthy and diseased state. They can help reducing the number of animal experiments, and hence support the 3R principle. This review discusses ALI cultures and co‐cultures derived from immortalized as well as primary cells, which are used to study the most common disorders of the respiratory tract, in terms of both pathophysiology and drug screening. The article displays ALI models used to simulate inflammatory lung diseases such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer, and viral infections. It also focuses on ALI cultures described in literature studying respiratory viruses such as SARS‐CoV‐2 causing the global Covid‐19 pandemic at the time of writing this review. Additionally, commercially available models of ALI cultures are presented. Ultimately, the aim of this review is to provide a detailed overview of ALI models currently available and to critically discuss them in the context of the most prevalent diseases of the respiratory tract.

Inhaled siRNA therapy has a unique potential for treatment of severe lung diseases, such as cystic fibrosis (CF). Nevertheless, a drug delivery system tackling lung barriers is mandatory to enhance gene silencing efficacy in the airway epithelium. We recently demonstrated that lipid-polymer hybrid nanoparticles (hNPs), comprising a poly(lactic-co-glycolic) acid (PLGA) core and a lipid shell of dipalmitoyl phosphatidylcholine (DPPC), may assist the transport of the nucleic acid cargo through mucus-covered human airway epithelium. To study in depth the potential of hNPs for siRNA delivery to the lungs and to investigate the hypothesized benefit of PEGylation, here, an siRNA pool against the nuclear factor-κB (siNFκB) was encapsulated inside hNPs, endowed with a non-PEGylated (DPPC) or a PEGylated (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) or DSPE-PEG) lipid shell. Resulting hNPs were tested for their stability profiles and transport properties in artificial CF mucus, mucus collected from CF cells, and sputum samples from a heterogeneous and representative set of CF patients. Initial information on hNP properties governing their interaction with airway mucus was acquired by small-angle X-ray scattering (SAXS) studies in artificial and cellular CF mucus. The diffusion profiles of hNPs through CF sputa suggested a crucial role of lung colonization of the corresponding donor patient, affecting the mucin type and content of the sample. Noteworthy, PEGylation did not boost mucus penetration in complex and sticky samples, such as CF sputa from patients with polymicrobial colonization. In parallel, in vitro cell uptake studies performed on mucus-lined Calu-3 cells grown at the air−liquid interface (ALI) confirmed the improved ability of non-PEGylated hNPs to overcome mucus and cellular lung barriers. Furthermore, effective in vitro NFκB gene silencing was achieved in LPS-stimulated 16HBE14o-cells. Overall, the results highlight the potential of non-PEGylated hNPs as carriers for pulmonary delivery of siRNA for local treatment of CF lung disease. Furthermore, this study provides a detailed understanding of how distinct models may provide different information on nanoparticle interaction with the mucus barrier.

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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

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To develop stable and inhalable dry powder formulations with long shelf life, polyplexes consisting of siRNA and a polyethylenimine (PEI)‐based block copolymer in presence of mannitol or trehalose are spray dried. The effect of inlet (T‐In) and outlet (T‐Out) temperature on the recovery of siRNA and adsorption effects within the tubing material are investigated. Choosing a low abrasion silicon tubing prevented siRNA loss due to adsorption. Mannitol and trehalose formulations preserved siRNA integrity regardless of excipient concentration and temperature at T‐Out below the siRNA melting temperature. Trehalose formulations allowed full siRNA recovery whereas mannitol formulations resulted in spray drying induced losses of ≈20% siRNA and of 50–60% polymer. Mannitol formulations showed optimal aerodynamic characteristics as confirmed by next generation impaction analysis based upon siRNA content. All spray dried formulations resulted in green fluorescent protein (GFP) silencing comparable or better than freshly prepared polyplexes. To test if the observed results could be transferred, formulations of siRNA and transferrin‐PEI conjugates are spray dried, characterized, and used to transfect primary human T cells ex vivo. Results confirmed successful silencing of the transcription factor GATA3 in primary CD4+ T cells with spray dried formulations as a potential treatment for severe asthma.

Polyethylenimine (PEI) is a highly efficient cationic polymer for nucleic acid delivery, and although it is commonly used in preclinical studies, its clinical application is limited because of concerns regarding its cytotoxicity. Poly(β-amino ester)s are a new group of biodegradable and biocompatible cationic polymers that can be used for siRNA delivery. In this study, we synthesized Boc-protected and deprotected poly(β-amino ester)s, P(BSpBAE) and P(SpBAE), respectively, based on spermine and 1,4-butanediol diacrylate to deliver siRNA. The polymers were synthesized by Michael addition in a step-growth polymerization and characterized via 1H NMR spectroscopy and size-exclusion chromatography (SEC). The polymers can encapsulate siRNA as determined by SYBR gold assays. Both polymers and polyplexes were biocompatible in vitro. Furthermore, the cellular uptake of P(BSpBAE) and P(SpBAE) polyplexes was more efficient than for branched PEI (25 kDa) polyplexes at the same N/P ratios. P(BSpBAE) polyplexes achieved 60% eGFP knockdown in vitro, which indicates that the Boc-protection can improve the siRNA delivery and gene silencing efficiency of PBAEs. P(BSpBAE) polyplexes and P(SpBAE) polyplexes showed different cellular uptake mechanisms, and P(BSpBAE) polyplexes demonstrated decreased endosomal entrapment, which could explain why P(BSpBAE) polyplexes more efficiently mediated gene silencing than P(SpBAE) polyplexes. Furthermore, transfection of an siRNA against mutated KRAS in KRAS-mutated lung cancer cells led to around 35% (P(BspBAE)) to 45% (P(SpBAE)) inhibition of KRAS expression and around 33% (P(SpBAE)) to 55% (P(BspBAE)) decreased motility in a migration assay. These results suggest that the newly developed spermine-based poly(β-amino ester)s are promising materials for therapeutic siRNA delivery.