Uptake, Efficacy, and Systemic Distribution of Naked, Inhaled Short Interfering RNA (siRNA) and Locked Nucleic Acid (LNA) Antisense

Abstract

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

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

“…Moreover, promising results were obtained upon inhalation of free siRNA in human clinical trials for the treatment of patients infected with respiratory syncytial virus [36]. However, recent publications have clearly demonstrated the benefit of nanocarriers for siRNA inhalation therapy [9,32,37]. This was confirmed by our work, as the aspiration of free siRNA in the bronchoalveolar lumen of BALB/c mice resulted in negligible siRNA internalization by rAM and hence also failed to elicit a gene silencing effect.…”

“…For instance, the cell membrane is impermeable for hydrophilic macromolecules like nucleic acids, precluding direct access of siRNA to the intracellular RNAi machinery. Moreover, it has been reported that free siRNA is more rapidly distributed to the systemic circulation upon local pulmonary application, which decreases the siRNA dose in the bronchoalveolar lumen [9]. To avoid systemic exposure and enhance cellular delivery of siRNA to target cells, siRNAs typically require formulation into lipid-or polymer-based drug carriers.…”

A microRNA network regulates expression and biosynthesis of wild-type and ΔF508 mutant cystic fibrosis transmembrane conductance regulator

“…Although transient, partial, and airway-specific delivery of an miR-138 mimic or anti-SIN3A siRNA might be therapeutic, the efficient delivery of siRNA or miR mimics to airway epithelia is inefficient with current technology (49,50). Further advancements in this field will likely lead to new clinical applications.…”

The Influence of Natural Pulmonary Surfactant on The Efficacy of siRNA-Loaded Dextran Nanogels

“…However, an efficient siRNA delivery vehicle should still overcome the various extra-and intracellular barriers encountered following inhalation therapy [27].…”