title: The delivery and evaluation of RNAi therapeutics for heterotopic ossification pathologies.
abstract: RNA interference (RNAi) is a powerful tool being used to develop therapies for pathologies caused by gene overexpression. Heterotopic ossification pathologies such as trauma-induced heterotopic ossification and fibrodysplasia ossificans progressiva may be treatable with an RNAi approach. However, there is a lack of consensus in literature regarding the delivery conditions and evaluation of RNAi therapeutics in these disease models. Here, we describe in vitro protocols for the delivery of polymer-based RNAi therapeutics as well as a streamlined strategy for the assessment of osteoblast lineage progression due to dysregulated bone morphogenetic protein signaling. This strategy focuses on the quantification of early-stage osteoblast transcription factors RUNX2 and OSX, followed by the measurement of alkaline phosphatase activity and late-stage matrix deposition.
authors.affiliation: Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA.
authors.forename: Arun R
authors.initials: AR
authors.lastname: Shrivats
authors.forename: Jeffrey O
authors.initials: JO
authors.lastname: Hollinger
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D020869:Gene Expression Profiling; D018014:Gene Transfer Techniques; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D009999:Ossification, Heterotopic / Q000628:therapy*; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D011108:Polymers; D034622:RNA Interference / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results; D013997:Time Factors
chemical_list: D015415:Biomarkers; D055415:Bone Morphogenetic Protein 4; D011108:Polymers; D034741:RNA, Small Interfering; D000469:Alkaline Phosphatase