骨肉瘤是青少年高发的骨恶性肿瘤，常伴发肺转移与肿瘤部位的骨破坏，预后极差。VEGFA对肿瘤微环境血管的生成以及肿瘤细胞自身的生长上起着重要作用，是治疗骨肉瘤的潜在药物靶点。CRISPR/Cas9系统可以精确地实现基因编辑，从源头上阻断VEGFA的表达，但最大瓶颈是缺乏肿瘤靶向性的递送系统。核酸适配子是可以与特定的细胞结合的单链寡核苷酸。我们筛选到了特异靶向骨肉瘤细胞的核酸适配子LC09，并制备了针对VEGFA基因编辑的CRISPR/Cas9的体内递送系统。体外实验发现LC09可促进CRISPR/Cas9在骨肉瘤细胞中的摄入，抑制VEGFA的内分泌和旁分泌通路。体内分布实验证实LC09对CRISPR/Cas9的骨肉瘤靶向递送能力。在本项目申请中，我们预期此系统可有效安全的抑制骨肉瘤的生长和肺转移，减少肿瘤血管生成和骨破坏，为CRISPR/Cas9在癌症治疗的临床转化提供实验依据。

Osteosarcoma (OS) is a highly aggressive pediatric cancer, characterized by distant metastasis (frequently spreads to the lungs) and local bone destruction. Vascular endothelial growth factor A (VEGFA), highly expressed in OS, not only acts as an autocrine survival factor for tumor cell themselves, but also contributes to angiogenesis within tumor microenvironment, thus making itself a promising therapeutic target for OS. CRISPR/Cas9 is a versatile genome editing technology and hold tremendous promise for development of new therapies. However, a major bottleneck to achieve the therapeutic potential of the CRISPR/Cas9 is the lack of in vivo targeted delivery systems. We screened an OS cell-specific aptamer (LC09) and developed LC09-functionalized lipopolymer encapsulating CRISPR/Cas9 plasmids encoding VEGFA-specific gRNA and Cas9. Our in vitro results demonstrated that the delivery system could facilitate the uptake of CRISPR/Cas9 in OS cells, and simultaneously restrained autocrine and paracrine functions of VEGFA signaling in OS cells. Our preliminary in vivo results showed that LC09 facilitated selective distribution of CRISPR/Cas9 in both orthotopic OS and pulmonary metastasis. In this project, we will further examine the in vivo effects of LC09-PPC-CRISPR/Cas9 on osteosarcoma growth and metastasis, angiogenesis and bone destruction. This project will provide an experimental-based strategy for clinical translation of CRISPR/Cas9.