title: Development of pre-clinical models for evaluating the therapeutic potential of candidate siRNA targeting STAT6.
abstract: Developing siRNA therapeutics poses technical challenges including appropriate molecular design and testing in suitable pre-clinical models. We previously detailed sequence-selection and modification strategies for siRNA candidates targeting STAT6. Here, we describe methodology that evaluates the suitability of candidate siRNA for respiratory administration. Chemically-modified siRNA exhibited similar inhibitory activity (IC50) against STAT6 in vitro compared to unmodified siRNA and apical exposure testing with Caco-2 cell monolayers showed modification was not associated with cellular toxicity. Use of a modified RNA extraction protocol improved the sensitivity of a PCR-based bio-analytical assay (lower limit of siRNA strand quantification  =  0.01 pg/µl) which was used to demonstrate that lung distribution profiles for both siRNAs were similar following intra-tracheal administration. However, after 6 hours, modified siRNA was detected in lung tissue at concentrations >1000-fold higher than unmodified siRNA. Evaluation in a rat model of allergic inflammation confirmed the persistence of modified siRNA in vivo, which was detectable in broncho-alveolar lavage (BAL) fluid, BAL cells and lung tissue samples, 72 hours after dosing. Based upon the concept of respiratory allergy as a single airway disease, we considered nasal delivery as a route for respiratory targeting, evaluating an intra-nasal exposure model that involved simple dosing followed by fine dissection of the nasal cavity. Notably, endogenous STAT6 expression was invariant throughout the nasal cavities and modified siRNA persisted for at least 3 days after administration. Coupled with our previous findings showing upregulated expression of inflammatory markers in nasal samples from asthmatics, these findings support the potential of intranasal siRNA delivery. In summary, we demonstrate the successful chemical modification of STAT6 targeting siRNA, which enhanced bio-availability without cellular toxicity or reduced efficacy. We have established a robust, sensitive method for determining siRNA bio-distribution in vivo, and developed a nasal model to aid evaluation. Further work is warranted.
authors.affiliation: College of Medicine, Swansea University, Swansea, United Kingdom ; Allerna Therapeutics Ltd, Swansea University, Swansea, United Kingdom.
authors.forename: Gareth D
authors.initials: GD
authors.lastname: Healey
authors.affiliation: Lockridge Pharmaceutical Consulting LLC, Westminster, Colorado, United States of America.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Lockridge
authors.affiliation: Zincyte Consulting, Denver, Colorado, United States of America.
authors.forename: Shawn
authors.initials: S
authors.lastname: Zinnen
authors.affiliation: College of Medicine, Swansea University, Swansea, United Kingdom.
authors.forename: Julian M
authors.initials: JM
authors.lastname: Hopkin
authors.affiliation: Sunapten Therapeutics, Kalamazoo, Michigan, United States of America.
authors.forename: Ivan
authors.initials: I
authors.lastname: Richards
authors.affiliation: College of Medicine, Swansea University, Swansea, United Kingdom.
authors.forename: William
authors.initials: W
authors.lastname: Walker
mesh_terms: D000281:Administration, Intranasal; D000485:Allergens / Q000276:immunology; D000818:Animals; D001249:Asthma / Q000235:genetics / Q000276:immunology / Q000628:therapy; D018938:Caco-2 Cells; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical; D005786:Gene Expression Regulation; D006801:Humans; D008168:Lung / Q000276:immunology / Q000378:metabolism; D008297:Male; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D051381:Rats; D050800:STAT6 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D000485:Allergens; D034741:RNA, Small Interfering; D050800:STAT6 Transcription Factor