Aptamer-Functionalized Delivery System for Osteogenic sirnas to Achieve Osteoblast- Specific RNA Interference for Bone Anabolic Therapy

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1 Aptamer-Functionalized Delivery System for Osteogenic sirnas to Achieve Osteoblast- Specific RNA Interference for Bone Anabolic Therapy Chao Liang 1,2, Baosheng Guo 1, Heng Wu 1, Lingqiang Zhang 2, Aiping Lu 1,3, Ge Zhang 1. 1 Joint Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, 2 State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China, 3 Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing, China. Disclosures: C. Liang: None. B. Guo: None. H. Wu: None. L. Zhang: None. A. Lu: None. G. Zhang: None. Introduction: Metabolic skeletal disorders associated with impaired bone formation (e.g., osteoporosis) remain major clinical challenges. RNA interference (RNAi) provides a promising approach for promoting osteoblastic bone formation to settle the above challenges. However, the bottleneck for translating RNAi is lack of cell-specific delivery systems for osteogenic sirnas. Previously, we developed a delivery system (AspSerSer)6-liposome which could target bone formation surface at tissue level and facilitate bone anabolic action of osteogenic Plekho1 sirnas in osteopenic rodents (Zhang et al. Nature Medicine 2012). However, due to lack of selectivity at cellular level (direct osteoblast-specific delivery), concerns about efficacy and safety could still not be adequately released, which is a major obstacle for clinical translation of RNAi-based bone anabolic strategy in metabolic skeletal disorders with impaired bone formation. In addition, other issues including mononuclear phagocyte system (MPS)-induced dose reduction, inefficient nanoparticle extravasation and detrimental accumulation in hepatocytes, also raise concerns about therapeutic efficacy and safety. Methods: Aptamers selected by cell-systematic evolution of ligands by exponential enrichment (cell- SELEX) with osteoblasts as target cells and hepatocytes together with peripheral blood mononuclear cells (PBMCs) as non-target cells may provide an approach to achieve osteoblast-specific delivery of osteogenic sirnas at cellular level. Non-target of hepatocytes and PBMCs will reduce the accumulation of sirnas in hepatocytes and MPS uptake of sirnas, respectively. In addition, lipid nanoparticles (LNPs) with small particle size (below 90 nm) and sufficient PEG shielding could serve as sirna delivery carriers to realize efficient extravasation and reduce MPS uptake of osteogenic sirnas. In our experiments, osteoblast-specific aptamer selected by cell-selex was conjugated to LNPs that encapsulated osteogenic Plekho1 sirna (Lu K, Nat Cell Bio 2008), i.e., Aptamer-LNPs-siRNA. In vitro evaluation of Aptamer-LNPssiRNA was conducted by cyto-tem, flow cytometry and confocal imaging in terms of physicochemical properties and biological characteristics. In vivo studies from biophotonic imaging, immunohistochemistry, laser capture microdissection (LCM) in combination with real-time PCR, microct and bone histomorphometry were performed to examine the selectivity and efficacy of Aptamer-LNPssiRNA, including tissue/cell-selective delivery, gene knockdown efficiency, dose-response pattern and bone anabolic action. Results: Using cell-selex, we screened an aptamer (termed as CH6 ) which targeted osteoblasts rather than hepatocytes and PBMCs. Surprisingly, the CH6 aptamer also recognized human osteoblasts rather than non-osteoblast cells (Figure 1). Then, we linked nuclease-resistance modified CH6 aptamer to LNPs

2 encapsulating osteogenic Plekho1 sirna, i.e., CH6-LNPs-siRNA, which had an average particle size of 84.0±5.3 nm and high encapsulation efficiency above 80%. CH6-LNPs-siRNA showed satisfactory serum stability and no significant cytotoxicity in vitro (Figure 2). In addition, CH6 aptamer facilitated the intracellular uptake of Plekho1 sirna (encapsulated in LNPs) and the subsequently gene knockdown in osteoblasts in vitro (Figure 2). Furthermore, in vivo biophotonic imaging and quantitative fluorescent analysis showed that CH6 facilitated the distribution of sirna in bone rather than in liver, kidney, brain, heart and spleen (Figure 2). Immunohistochemistry results showed that CH6 achieved in vivo osteoblastspecific delivery of Plekho1 sirna (Figure 2). LCM in combination with real-time PCR results showed that CH6 facilitated in vivo osteoblast-specific gene silencing (Figure 3). In addition, dose-response experiment showed that CH6-LNPs-siRNA achieved almost 80% gene knockdown efficiency at the sirna dose of 1.0 mg/kg and subsequently maintained 12 days for over 50% gene silencing (Figure 3). MicroCT and bone histomorphometric analysis confirmed that CH6 facilitated bone formation, leading to improved bone micro-architecture and increased bone mass in ovariectomized rats (Figure 3). Discussion: Here, we obtained an aptamer (CH6) with rat osteoblasts as target cells and rat hepatocytes and PBMCs as non-target cells. CH6 aptamer could also bind with human osteoblasts with a potency and specificity similar to that observed in rat osteoblasts, implying the great translational potential of CH6 aptamer in clinical application. We further developed CH6-functionalized LNPs encapsulating osteogenic Plekho1 sirna, i.e., CH6-LNPs-siRNA. The in vitro data suggested that the linked CH6 to the LNPs-based delivery system selectively targeted osteoblasts and subsequently facilitated osteoblast-specific localization of the encapsulated Plekho1 sirna. Consistently, the in vivo data from biophotonic imaging, immunohistochemistry examination, real-time PCR, microct and bone histomorphometric analysis confirmed that CH6 facilitated skeleton tissue/osteoblast-specific delivery of Plekho1 sirna and long persistence of gene knockdown, leading to promoted bone formation, improved bone microarchitecture and increased bone mass in osteopenic rats. Significance: To our knowledge, the osteoblast-specific aptamer-functionalized LNPs is the first one to advance selectivity of targeted delivery for osteogenic sirnas from tissue level toward cellular level for accommodating clinical translation of RNAi-based bone anabolic strategy.

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6 ORS 2015 Annual Meeting Paper No: 0030