适配体介导的表达多个siRNA的条件复制型腺病毒在恶性脑胶质瘤靶向治疗的实验研究

Replication-deficient adenoviral vector has a unique advantage in cancer gene therapy because of its merits with high-capacity, high-titer, no chromosomal integration and low pathogenicity on human. However, it also has some shortcomings that the adenovirus cannot be amplified in tumor cells and has lower transduction efficiencies on tumor cells, which limit its therapeutic efficacy on malignant gliomas. For enhancing effective glioblastoma theray, we for the first time developed a novel vector CRAd5-Sp-Hexon-BAP-siRNAs-aptamer by the following strategies: 1.To construct a conditionally replicative adenoviral vector (CRAd5-Sp) that utilizes the survivin promoter to improve its replicate efficiency for glioblastoma. 2. To insert the biotin-binding peptide (BAP) into the hypervariable region (HVR) 5 of the hexon, then the vector could be targeted to gliomas by the interaction between BAP present in viral vector and avidin- biotinylated aptamers .3. To insert an RFP-intron splicing system expressing multiple siRNAs into the region between the fiber and E4.The obtained novel conditionally replicating adenovirus CRAd5-Sp-Hexon-BAP-siRNAs-aptamer could enhance its transduction efficiency and cytotoxicity for gliomas with reduced cytotoxicity on normal cell. The novel vector offers a new option for glioblastoma multiforme therapy and other tumors.

复制缺陷型腺病毒载体由于容量大、滴度高及无致瘤性等优点在肿瘤基因治疗中具有独特的优势，但也存在肿瘤细胞中无法复制和感染率低等不足，因此复制缺陷型腺病毒载体的肿瘤治疗效果并不十分理想。本研究拟从以下三个方面对腺病毒载体进行改造：1.采用肿瘤特异性Survivin启动子，构建条件复制型腺病毒载体CRAd5-Sp；2.构建Hexon蛋白经BAP修饰的CRAd5-Sp-Hexon-BAP，利用biotin-avidin的高亲和力，结合经筛选的生物素化的适配体，从而介导靶向恶性脑胶质瘤；3.在病毒载体fiber与E4之间插入可表达多个具有协同治疗作用的siRNAs的内含子-siRNAs系统。由此获得新型的条件复制型腺病毒载体CRAd5-Sp-Hexon-BAP-siRNAs-aptamer可提高自身对恶性脑胶质瘤的感染效率和杀伤效率，降低对正常细胞的影响，为恶性脑胶质瘤及其它肿瘤的治疗开辟新的途径。

恶性脑胶质母细胞瘤血管生长丰富，侵蚀性强，与脑组织无明显分界，手术难以彻底切除、易复发、生存期短、死亡率高，是严重危害人类健康的肿瘤疾病之一。目前对此类疾病除化疗及放疗外，还没有找到有效的对策和治疗手段，因此探索新的治疗方法显得极为重要。本研究通过构建了Hexon蛋白经BAP修饰的腺病毒载体，同时筛选针对恶性脑胶质瘤的生物素化的适配体，利用biotin-avidin的高亲和力结合，形成适配体介导的新型腺病毒。通过体外实验证明，适配体介导的新型腺病毒相比对照病毒组，对恶性脑胶质瘤的感染率提高4-5倍，并对正常细胞没有影响，说明适配体可以显著提高腺病毒对肿瘤细胞的感染效率，本研究为恶性脑胶质瘤的治疗开辟新的途径。项目执行期间，发表在J Biotechnol等国际知名杂志发表SCI文章4篇，核心文章4篇，同时获得国家授权发明专利1项。本研究实现了在Hexon经BAP修饰的腺病毒载体骨架病毒骨架的基础上，结合不同适配体，靶向多种肿瘤细胞的通用模式。这种通用性模式为腺病毒的肿瘤基因治疗提供有效的手段也具备了非常好的应用前景和经济性。