(Asp-Ser-Ser)6 facilitates bone anabolic siRNA specifically targeting osteogenic cells in aged osteoporotic rats

1, 2Guo, B; 3Zhang, B; 4Wang, J; 1, 5Liang, C; 1Wu, H; 1Yang, Z; 6Chow, A; 2Hou, Z; 2Huang, Y; 5He, F; 7Qin, L; 5Zhang, L; 1Bian, Z; 1Lu, A; 1, 2Zhang, G

1School of Chinese Medicine, Hong Kong Baptist University, HK, Hong Kong, 2Jinan University, Guangzhou, China, 3The Chinese University of Hong Kong, HK, Hong Kong, 4South University of Science and Technology of China, Shenzhen, China, 5Beijing Institute of Radiation Medicine, Beijing, China, 6The Chinese University of Hong Kong, HK, China, 7The Chinese University of Hong Kong, HK, Hong Kong


Introduction: (Asp-Ser-Ser)6 is a newly identified peptide selectively targeting osteogenic cells via specifically approaching bone formation surface, which has been validated as a target moiety for targeted delivery of osteogenic siRNA (e.g. CKIP-1 siRNA) encapsulated within liposome in adult healthy rodents (Zhang et al, 2010). So, the target moiety could facilitate osteogenic siRNA targeting osteogenic cells to achieve RNAi-based bone anabolic strategy in reversing established osteoporosis. However, the targeted delivery facilitated by the newly identified peptide (Asp-Ser-Ser)6 has not been investigated in rodents with established osteoporosis, e.g. ovariectomy-induced osteoporosis. In this study, we will examine the distribution and knockdown efficiency of the CKIP-1 siRNA delivered by the (Asp-Ser-Ser)6-Liposome at both tissue level (skeletal tissue and non-skeletal tissues) and cell level (osteogenic cells and non-osteogenic cells ) in aged ovariectomized rats.

Methods: Twenty six-month-old female Sprague-Dawley rats were ovariectomized and left untreated for 12 months until 18-mohtn-old. Thereafter, all the rats were intravenously injected with CKIP-1 siRNA delivered with liposome (Liposome-siRNA Group, n=10) or CKIP-1 siRNA delivered with (Asp-Ser-Ser)6-Liposome ((Asp-Ser-Ser)6-Liposome siRNA Group, n=10). The CKIP-1 siRNA was labeled with FAM (fluorescein amidite). All the rats in each group were sacrificed twelve hours later after the administration. To evaluate the distribution and knockdown efficiency of CKIP-1 siRNA at tissue level, the existence of the fluorescence-labeled siRNA in skeletal tissue (left femur) and non-skeletal tissues (Heart, Liver, Spleen, Lung, Kidney) was visualized by IVIS biophotonic imaging system and the accumulation was quantified by a microplate reader system, respectively. CKIP-1 mRNA level in each tissue was analyzed using real-time PCR for knockdown efficiency. To examine the distribution and knockdown efficiency of CKIP-1 siRNA at cell level in skeletal tissue, the right femur was dissected and then decalcified. After that, the cryosections from the decalcified distal femur were subjected to immunostaining for co-localization of siRNA signal and osteocalcin. Thereafter, laser capture micro-dissection (LCM) in combination with Q-PCR analysis was performed for CKIP-1 mRNA level in osteogenic/non-osteogenic cells as determined by osteocalcin-positive staining.

Results: Biophotonic imaging demonstrated that the intensity of the intraosseous fluorescence signal was significantly higher in (Asp-Ser-Ser)6-Liposome-siRNA group than that in Liposome-siRNA group, whereas the intensities of the fluorescence signal in those non-skeletal tissues were all significantly lower in (Asp-Ser-Ser)6-Liposome-siRNA group than that in Liposome-siRNA group (Figure 1A). The quantification data from the microplate reader system was in accordance with that from biophotonic imaging (Figure 1B). Consistently, the knockdown efficiency in the skeletal tissue was also significantly higher in (Asp-Ser-Ser)6-Liposome-siRNA group than that in Liposome-siRNA group, whereas that was significantly lower in those non-skeletal tissues in (Asp-Ser-Ser)6-Liposome-siRNA group than that in Liposome-siRNA group (data not shown). The immunostaining data showed numerous instances of co-localization of the labeled siRNA with osteocalcin-positive (OCN+) cells in (Asp-Ser-Ser)6-Liposome-siRNA group than that in Liposome-siRNA group (Figure 2). The knockdown efficiency of CKIP-1 siRNA in OCN+ cells was significantly higher when compared to that in Osteocalcin-negative (OCN-) cells in (Asp-Ser-Ser)6-Liposome-siRNA group, whereas no difference was found between OCN+ cells and OCN- cells in Liposome-siRNA group (Figure 3)

Discussion: (Asp-Ser-Ser)6 could facilitate bone anabolic siRNA specifically targeting osteogenic cells in aged ovariectomized rats for translation of RNAi-based bone anabolic strategy.

Significance: In this study, the (Asp-Ser-Ser)6 has been validated in osteoporotic rats for bone-targeted anabolic siRNA delivery which would facilitate the tranlation study of anabolic siRNA in osteoporosis treatment.

Acknowledgements: This study was supported by both Hong Kong General Research Fund (479111 and 478312) and CUHK-High Promise Initiatives Scheme B (1902063).

References: Zhang G, et al. 2012 A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy. Nat Med 18(2):307-14.

Figure 1 Tissue distribution analysis by biophotonic imaging system (A) and quantitative analysis by microplate reader system (B) for labeled siRNA in Liposome-siRNA group and (Asp-Ser-Ser)6-Liposome-siRNA group. *P < 0.05 vs Liposome-siRNA group. *P < 0.05 vs. Liposome-siRNA group.
Figure 2 Co-localization of FAM-labeled siRNA (green, arrow indicated), osteocalcin-positive staining cells (red, arrow indicated) and nucleus staining with DAPI (blue, arrow indicated) in distal femur of rats from Liposome-siRNA group and (Asp-Ser-Ser)6-Liposome-siRNA group.
Figure 3 Osteocalcin-positive staining cells in cryosections from distal femur were determined as osteogenic cells (left, arrow indicated). These cells in the adjacent cryosections were stained with fast red (middle, arrow indicated) and dissected by LCM (right, arrow indicated) (A) and the knockdown efficiency of CKIP-1 mRNA in osteogenic or non-osteogenic cells was examined by Q-PCR analysis (B). Scale bar= 50 µm. * P < 0.05 for OCN- vs. OCN+