Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo

Griesenbach, Uta; Kitson, Christopher; Garcia, Escudero Sara; Farley, Raymond; Singh, Charanjit; Somerton, Luci; Painter, Hazel; Smith, R.; Gill, Deborah R.; Hyde, Stephen C.; Chow, Yu‐Hua; Hu, Jim; Gray, Michael A.; Edbrooke, Mark R.; Ogilvie, Varrie; MacGregor, Gordon; Scheule, Ronald K.; Cheng, Seng H.; Caplen, Natasha J.; Alton, Eric W.F.W.

doi:10.1186/1465-9921-7-26

Cited by 59 publications

(57 citation statements)

References 32 publications

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“…However, the pseudostratified epithelium presents significant barriers to the delivery of nucleic acid based reagents such as plasmids (17, 65), doublestranded oligonucleotides (9,23,36,50), and single-stranded oligonucleotides (9, 23). It is evident that physical barriers, airway secretions, and host defense mechanisms (9,17,23,49,62) significantly hinder the delivery and efficacy of the siRNA reagents. The mucosal surface of the airways and the alveoli elicits immunogenic responses to RNA interference (RNAi) oligonucleotides that confound results (13,31,35,45,54), and some animal studies attempting to deliver siRNAs to the respiratory tract have reported little efficacy (23).…”

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Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

Ramachandran

Krishnamurthy

Jacobi

et al. 2013

American Journal of Physiology-Lung Cellular and Molecular Phys

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Ramachandran S, Krishnamurthy S, Jacobi AM, WohlfordLenane C, Behlke MA, Davidson BL, McCray PB, Jr. Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro. Am J Physiol Lung Cell Mol Physiol 305: L23-L32, 2013. First published April 26, 2013 doi:10.1152/ajplung.00426.2012.-Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-totransfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1-3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Cl Ϫ conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses. primary airway epithelium; transfection; gene silencing; RNA interference; siRNA; air-liquid interface; porcine airway epithelial cells SMALL INTERFERING RNAs (siRNA) mediate posttranscriptional inhibition of targeted genes, offering a novel approach to achieve specific silencing in the airway epithelium. These approaches have broad applications for studies of cell biology, disease pathogenesis, and therapeutics. Primary cultures of airway epithelia have emerged as a powerful model to study epithelial cell biology and the impact of diseases on tissue function (18,22,39). Cells grown at the air-liquid interface (ALI) form a polarized, pseudostratified columnar epithelium that closely resembles the morphology of the in vivo surface epithelium of the conducting airways (2,18,24,33,63,64), providing an opportunity to study cell biology, disease pathogenesis, and treatments (66).Histological studies have shown that ALI cultures of primary airways grown for 4 wk or more form a well-differentiated epithelium resembling the in vivo architecture (12, 56). However, another study has observed that airway epithelia...

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confidence: 99%

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confidence: 99%

Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

Ramachandran

Krishnamurthy

Jacobi

et al. 2013

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…A multitude of novel siRNA nanocarriers has already been tested in vitro. Still, to date only a limited number of nanoparticles have been evaluated for local in vivo siRNA delivery to rAM, with variable success [14][15][16].…”

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confidence: 99%

Hybrid pulmonary surfactant-coated nanogels mediate efficient in vivo delivery of siRNA to murine alveolar macrophages

Backer

Naessens

Koker

et al. 2015

Journal of Controlled Release

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The local delivery of small interfering RNA (siRNA) to the lungs may provide a therapeutic solution to a range of pulmonary disorders. Resident alveolar macrophages (rAM) in the bronchoalveolar lumen play a critical role in lung inflammatory responses and therefore constitute a particularly attractive target for siRNA therapeutics. However, achieving efficient gene silencing in the lung while avoiding pulmonary toxicity requires appropriate formulation of siRNA in functional nanocarriers. In this study, we evaluated pulmonary surfactant-coated dextran nanogels for the delivery of siRNA to rAM upon pharyngeal aspiration in BALB/c mice. Both the surfactant-coated and uncoated nanogels achieved high levels of siRNA uptake in rAM, yet only the surfactant-coated formulation could significantly reduce gene expression on the protein level. Surfactant-coated nanogels induced a profound downregulation of target mRNA levels, reaching 70% knockdown with ~1 mg kg -1 siRNA dose. In addition, only mild acute pro-inflammatory cytokine and chemokine responses were detected one day after nanoparticle aspiration, accompanied by a moderate neutrophil infiltration in the bronchoalveolar lumen. The latter could be substantially reduced by removal of excess surfactant from the formulation. Overall, our hybrid core-shell nanoparticles have demonstrated safe and effective siRNA delivery to rAM, providing a new therapeutic approach for treatment of inflammatory pathologies in the lung.

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“…Firstly, as PXS TPI1100 is administered via inhalation, it is delivered directly to the intended site of action of the lung (Ali et al 2001;Duan et al 2005;Gauvreau et al 2008;Guimond et al 2008) where the drug can enter target cells directly (Zhang et al 2004;Griesenbach et al 2006) thus potentially reducing total dose as compared to orally-available treatments. A further advantage of pulmonary administration of AON is that they are principally www.intechopen.com A Multi-Targeted Antisense Oligonucleoitde-Based Therapy Directed at Phosphodiesterases 4 and 7 for COPD 449 metabolized in the lung with very limited systemic delivery after inhalation (Templin et al 2000;Ali et al 2001;Guimond et al 2008) which leads to reduced systemic bioavailability of the drug.…”

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confidence: 99%

A Multi-Targeted Antisense Oligonucleotide-Based Therapy Directed at Phosphodiesterases 4 and 7 for COPD

Séguin¹,

Ferrari²

2012

Chronic Obstructive Pulmonary Disease - Current Concepts and Practice

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Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo

Cited by 59 publications

References 32 publications

Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

Hybrid pulmonary surfactant-coated nanogels mediate efficient in vivo delivery of siRNA to murine alveolar macrophages

A Multi-Targeted Antisense Oligonucleotide-Based Therapy Directed at Phosphodiesterases 4 and 7 for COPD

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