NEW YORK (GenomeWeb) – While delivery has long been the main hurdle for siRNA therapeutics, certain cell types including osteoblasts have proven especially difficult to target with the gene-silencing molecules. But in a new study, researchers from Hong Kong Baptist University have demonstrated an aptamer-based approach that promises to overcome this limitation, opening the door to RNAi treatments for a variety of bone-formation disorders.
In the report, which appeared in Nature Medicine this week, focused on silencing a gene called Plekho1, which is a known intracellular negative regulator of bone formation that does not affect bone resorption.
In an earlier study, the research team showed that Plekho1-targeting siRNAs could be delivered to bone-forming cells — but those related to bone resorption — near the surface of bones using a targeting system involving DOTAP-based cationic liposomes attached to six repetitive sequences of aspartate, serine, and serine (AspSerSer).
Systemic delivery of the siRNAs in rats resulted in the selective enrichment in osteogenic cells and the subsequent depletion of Plekho1, and markedly promoted bone formation in both healthy and osteoporotic animals, according to that study.
However, that delivery system was not specific to osteoblasts at the cellular level, raising concerns about potential adverse effects stemming from Plekho1 silencing in other non-osteoblasts near the bone-formation surface, including endothelial cells and lymphocytes, the Hong Kong Baptist University scientists wrote in their latest paper.
The non-specificity of this first delivery approach could also result in problems with mononuclear phagocyte system-induced dose reduction, inefficient nanoparticle extravasation caused by large particle size, and detrimental hepatocyte accumulation of siRNAs, they added.
To address this issue, the investigators looked to combine lipid nanoparticles (LNPs), which can protect nucleic acid payloads and carry them into cells, with aptamers, which are single-stranded oligos that can be designed to target only certain cell-surface receptors.
They began by cell-based systematic evolution of ligands by exponential enrichment, or cell-SELEX, to select osteoblast-specific aptamers. "By performing positive selection with osteoblasts and negative selection with hepatocytes and peripheral blood mononuclear cells (PBMCs), we aimed to screen an aptamer that could achieve direct osteoblast-specific delivery of osteogenic siRNAs and minimal hepatocyte and PBMC accumulation of osteogenic siRNAs," the team wrote in Nature Medicine.
The scientists selected the aptamer CH6, which they conjugated to lipid nanoparticles encapsulating Plekho1 siRNAs. The molecules were then tested in vitro and in vivo in osteopenic and healthy rats.
They found that CH6 facilitated osteoblast-selective uptake of Plekho1 siRNA, mainly via macropinocytosis, in cell culture experiments. When administered intravenously into the rodents, the molecules triggered osteoblast-specific Plekho1 gene silencing, which promoted bone formation, improved bone microarchitecture, increased bone mass and enhanced mechanical properties in both groups of animals.
These effects, the researchers noted, were more pronounced than with their AspSerSer-liposome formulation — something they stated could be related to the delivery mechanisms behind each approach.
"The AspSerSer moiety was designed to target the physiochemical features of the bone formation surface, and as such it targeted osteoblasts at a tissue level," they wrote. "In contrast, the CH6 aptamer was selected to directly target osteoblasts at the cellular level, which is more specific and efficient."
Further, the macropinocytosis induced by the CH6 aptamer may also explain the high gene knockdown efficiency and enhanced bone anabolic action of the newer molecules.
Overall, the Hong Kong Baptist University team wrote, the findings indicate that osteoblast-specific aptamer-functionalized LNPs may "act as a new RNAi-based bone anabolic strategy, advancing the targeted delivery selectivity of osteogenic siRNAs from the tissue level to the cellular level."