The delivery and evaluation of RNAi therapeutics for heterotopic ossification pathologies.

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.