靶向破骨细胞核酸递送系统包裹微小RNA拮抗剂抑制骨吸收的转化研究

Skeletal diseases induced by osteoclast dysfunction, e.g. post-menopausal osteoporosis, are still great clinical challenges. Emerging evidences indicate that osteoclast dysfunction could be attributed to dysregulated microRNAs (miRNAs) within osteoclasts. The miRNA modulators (microRNA antagomir / agomir) have been widely employed to modulate intracellular miRNAs. Recently, several delivery systems have been designed to facilitate miRNA modulators targeting bone at tissue / organ level. However, systemic administration of microRNA modulators with those bone-targeted delivery systems still require large therapeutic dose, which may bring about high risk for off-target effects. Therefore, it is highly desirable to develop an osteoclast-specific delivery system for miRNA modulators at cellular level in vivo. .It is known that the osteoclasts are dominantly recruited at the niches around the bone resportion surfaces characterized by highly crystallized hydroxyapatite. In our published data, the applicant has demonstrated that D-Asp8 peptide favorably binds to highly crystallized hydroxyapatite, implying its potential as osteoclast-targeting moiety (Zhang G, Guo BS et al. Nature Medicine 2012). Therefore, the applicant conjugated D-Asp8 peptide with liposome and subsequently encapsulated antagomir-148a (a miRNA modulator suppressing osteoclastogenesis and inhibiting bone resorption), i.e. (D-Asp8)-liposome-antagomir-148a. After then, the applicant characterized the (D-Asp8)-liposome-antagomir-148a (Dang L, Guo BS et al. Molecular Therapy Supplement 2014). Thus, the applicant raised the hypothesis that D-Asp8 could facilitate antagomir-148a (encapsulated in liposome) specifically targeting osteoclast for therapeutic modulation of miR-148a to inhibit bone resorption. .To test the above hypothesis, an osteoporotic mouse model induced by ovariectomy will be used in our study. The applicants intend (1) To investigate the distribution and knockdown efficiency of (D-Asp8)-liposome-antagomir-148a at cellular level; (2) To investigate the mechanism of (D-Asp8)-liposome specific targeting osteoclasts. (3) To determine the inhibitory effects of (D-Asp8)-liposome-antagomir-148a on elevated bone resorption induced by ovariectomy; (4) To investigate whether pre-treatment of D-Asp8 could abolish osteoclast-specific delivery of (D-Asp8)-liposome-antagomir-148a and subsequently attenuate its inhibitory effects on elevated bone resorption induced by ovariectomy. (5) To examine the hepatic/renal toxicity and immune response of (D-Asp8)-liposome-antagomir-148a after single / multiple dose(s) injections in ovariectomized (OVX) mice..The current proposal would provide a novel osteoclast-specific delivery system for miRNA modulators to update the targeted delivery from tissue / organ level toward cellular level. It could facilitate clinical translation of miRNA modulators in treating those skeletal diseases induced by osteoclast dysfunction.

微小RNA拮抗剂能够调控破骨细胞功能异常，由于缺乏靶向破骨细胞的核酸递送系统，全身注射微小RNA拮抗剂需要高剂量并会引起副作用。申请人发现八个连续D型天冬氨酸多肽D-Asp8具有破骨细胞靶向潜能。 因此，将D-Asp8与脂质体liposome链接而形成新型靶向破骨细胞的核酸递送系统(D-Asp8)-liposome，并初步鉴定其理化性质。本项目中，申请人拟利用 (D-Asp8)-liposome包裹antagomiR-148a（一种能够抑制破骨细胞功能的微小RNA拮抗剂）通过静默破骨细胞内miR-148a调控破骨细胞功能进而抑制骨吸收。并应用去势小鼠骨质疏松模型进一步评价(D-Asp8)-liposome包裹antagomiR-148a的破骨细胞靶向性、静默效率、靶向机理、治疗潜能及肝肾毒性。从而为临床治疗破骨细胞功能障碍导致的骨代谢疾病提供行之有效的干预治。

破骨细胞功能障碍导致的骨代谢疾病是临床骨科治疗的巨大挑战。微小RNA拮抗剂能够调控破骨细胞功能异常，由于缺乏靶向破骨细胞的核酸递送系统，全身注射微小RNA拮抗剂需要高剂量并会引起副作用。申请人发现八个连续D型天冬氨酸多肽D-Asp8具有破骨细胞靶向潜能。 因此，将D-Asp8与脂质体liposome链接而形成新型靶向破骨细胞的核酸递送系统(D-Asp8)-liposome，并初步鉴定其理化性质。本研究过程项目中，申请人利用 (D-Asp8)-liposome包裹antagomiR-148a（一种能够抑制破骨细胞功能的微小RNA拮抗剂）通过静默 破骨细胞内miR-148a调控破骨细胞功能进而抑制骨吸收。并应用去势小鼠骨质疏松模型进一步 评价(D-Asp8)-liposome包裹antagomiR-148a的破骨细胞靶向性、静默效率、靶向机理、治疗 潜能及肝肾毒性。在项目研究结果证实：（1）罗丹明标记的(D-Asp8)-liposome包裹antagomiR-148a，即：Rhodamine B-(D-Asp8)-liposome-antagomiR-148a能够特异性静默去势小鼠骨破骨细胞的miR-148a的表达；（2）(D-Asp8)-liposome包裹的antagomiR-148a能够抑制去势小鼠骨吸收亢进；（3）(D-Asp8)-liposome预处理对(D-Asp8)-liposome包裹的antagomiR-148a的能够抑制去势小鼠骨吸收亢进，从而进一步确证(D-Asp8)-liposome靶向破骨细胞的机理；（4）体内注射(D-Asp8)-liposome包裹的antagomiR-148a不会引起肝肾毒性，具有很好安全性和肝肾毒性。从而为临床治疗破骨细胞功能障碍导致的骨代谢疾病提供行之有效的干预治 。