核酸适配子功能化的脂质聚合物肿瘤靶向递送CRISPR/Cas9用于基因编辑VEGFA治疗骨肉瘤

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.

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。

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。由于骨肉瘤内血管丰富表达大量的VEGFA，通过利用CRISPR/Cas9系统从基因层面抑制VEGFA的表达，可以从根源上抑制肿瘤微环境血管的生成以及肿瘤细胞自身的生长。CRISPR/Cas9系统的最大瓶颈是缺乏肿瘤靶向性的递送系统。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。在本项目中，我们进一步验证了此系统在小鼠原位骨肉瘤模型中的抗肿瘤抗转移活性、对肿瘤内部血管新生和骨破坏的影响，以及整个系统的体内安全性评价。我们发现我们开发的骨肉瘤适配子功能化的CRISPR/Cas9系统可以有效的抑制骨肉瘤的生长和肺转移，显著的减少肿瘤血管生成和肿瘤导致的骨破坏。同时，在有效治疗剂量所在的浓度范围内对小鼠的肝脏、肾脏及血液都没有显著的毒性。此课题的结果为适配子功能化的CRISPR/Cas9递送系统在癌症治疗的临床转化提供了实验依据。