A Circular Aptamer-ASO Chimera for Activating DLX5-Regulated Osteogenesis through Targeted Inhibition of STAT5A Expression

Therapies that stimulate DLX5-driven osteogenesis in bone-marrow-derived mesenchymal stem cells (BMSCs) with bone morphogenetic proteins (BMPs) potently accelerate the healing of delayed union and nonunion fractures, but their superior osteoinductive activity is often offset by severe adverse effects. To provide a safer and more effective alternative, we engineered a circular aptamer-antisense oligonucleotide chimera (CircApt-ASO) to activate DLX5-regulated osteogenesis by silencing STAT5A, a key negative regulator of DLX5. CircApt-ASO utilizes a transferrin receptor 1 (TFR1)-binding aptamer, enabling both specific nanoaffinity targeting of BMSCs and efficient intracellular delivery of the anti-STAT5A ASO. Compared with chemically modified linear aptamer-ASO chimeras, CircApt-ASO chimeras exhibit superior biostability and more robust STAT5A gene silencing but negligible cytotoxicity relative to liposome-based ASO delivery methods. Importantly, we demonstrated that CircApt-ASO drastically activated the expression of DLX5 and its downstream osteogenesis-related genes in dose- and time-dependent manners, leading to markedly enhanced osteogenic differentiation. The high stability, potent osteoinductive activity, and minimal cytotoxicity of CircApt-ASO highlight its strong therapeutic potential for promoting bone regeneration in conditions such as delayed union and nonunion fractures.