Aptamer-functionalized osteoblast-targeting delivery system for osteogenic siRNAs to achieve cell-specific RNA interference for bone anabolic therapy

Metabolic skeletal disorders associated with impaired bone formation are major clinical challenges. RNA interference (RNAi) for promoting osteoblastic bone formation is a promising approach to the challenges. The bottleneck for translating RNAi with efficiency and safety to clinical bone anabolic strategy is to seek targeted delivery systems for osteogenic siRNAs. The applicants have identified (AspSerSer)6 as a target moiety specifically approaching bone formation surface and further validated that (AspSerSer)6 could facilitate osteogenic siRNAs targeting osteogenic cells to promote bone formation in rodents (Zhang G. Nature Med 2012). However, the limitation of the specificity of the targeted delivery by (AspSerSer)6-liposome is not at cellular level but at tissue level, i.e. bone formation surface, which still cannot substantiality release concerns on efficacy and safety mainly due to lack of direct osteoblast-specific delivery. In addition, mononuclear phagocyte system (MPS)-induced loss of injected dose, inefficiency for large particle size to extravasate from bony sinusoids (< 100nm) and detrimental accumulation in hepatocytes should be also considered.

Aptamers are single-stranded oligonucleotides which can bind to target cells specifically by distinct tertiary structures even without knowledge of characteristic protein profiles on cell surface. Thus, aptamer, as targeting ligand, may provide an approach to achieve osteoblast-specific delivery by targeting osteoblasts but not hepatocytes and peripheral blood cells (PBCs). Non-target of hepatocytes and PBCs will facilitate reducing siRNA accumulation in liver and escaping from MPS uptake, respectively. Meanwhile, lipid nanoparticles (LNPs) could be made with a small particle size below 90 nm. Polyethylene glycol (PEG) can also efficiently prevent LNPs from opsonizing and subsequent uptake by MPS. So, LNPs with sufficient PEG shielding could serve as carriers for siRNAs to realize efficient extravasation from fenestrated capillaries to osteoblast and escape from MPS. Thus, aptamer-functionalized LNPs may be used as an efficient and safe delivery system for osteogenic siRNAs.

Using cell-SELEX technology, the applicants obtained an aptamers L6 (named according to its sequence code) which could target osteoblasts but not hepatocytes and PBCs in vitro (Liang C et al. 2012a). Thereafter, the applicants linked L6 aptamer to the surface of the PEG-modified LNPs encapsulating osteogenic siRNA, i.e. L6-LNPs-siRNA, which had an average particle size of 84.0±5.3 nm and high encapsulation efficiency above 80% (Wu H et al. 2012). L6-LNPs-siRNA showed satisfactory serum stability and no significant cytotoxicity in vitro. Further, osteoblast-selectivity of L6 aptamer did not alter after its linkage to LNPs-siRNA in vitro. L6 facilitated the intracellular uptake of the siRNA (encapsulated in LNPs) and subsequently gene knockdown in osteoblast other than in hepatocyte and PBC in vitro. Moreover, L6 aptamer facilitated the distribution of the siRNAs in bone and reducing their accumulation in liver in normal rats. We hypothesize that L6 aptamer could facilitate selectively targeting osteoblast for osteogenic siRNAs (encapsulated in LNPs) and subsequently promoting bone formation in established osteoporotic rodents.

The following specific aims will be achieved to test the above hypothesis in ovariectomy-induced osteoporotic rats treated with the L6-LNPs-siRNA using the established protocols (Zhang G. Nature Med 2012; Wang X, et al. Nature Med 2013; Reyes et al. 2010): (1) To examine the L6-mediated changes in distribution of the osteogenic siRNA at cellular level; (2) To test the L6-mediated changes in gene knockdown efficiency of the siRNA at cellular level; (3) To determine the L6-mediated changes in dose-response pattern and persistence of gene silencing; (4) To evaluate the L6-mediated changes in bone histomorphometric parameters/bone architecture/bone mass/bone mechanical properties. (5) To detect the L6-mediated changes in liver-related toxicity. (6) To investigate the mechanism of the L6-mediated siRNA cellular uptake and sub-cellular trafficking.

The current proposal would provide a novel aptamer-functionalized osteoblast-targeting delivery system for osteogenic siRNAs to update the targeted delivery from tissue level toward cellular level for accommodating translation of RNAi-based clinical bone anabolic strategy in efficiency and safety to metabolic skeletal disorders associated with impaired bone formation.