新型双级脑靶向溶瘤腺病毒载体设计及其治疗恶性胶质瘤的研究

Malignant glioma is the most common form of primary brain cancer and carries an extremely high recurrence rate and mortality rate. The dismal clinical context of advanced-grade glioma poses an urgent need for the development of novel and effective antiglioma therapies. Among many novel experimental therapeutic strategies that are currently being tested, oncolytic adenovirus (Ad) mediated gene therapy represents a potentially effective approach for glioma therapy. Besides the invasive surgical procedures upon vector administration into the brain, poor spreading of the Ad vectors in tumor and brain parenchyma have limited the development of gene therapy strategies for glioma. Therefore, development of Ad vectors suitable for overcoming the blood-brain barrier (BBB) non-invasively upon systemic administration has been suggested. However, there are still many limitations to the further use of adenoviral vector in the human body, such as the pre-existing anti-vector immunity, in vivo liver enrichment by intravenous administration, and the key problem how to across the BBB and target to tumor cells. In the present study, we will design new engineering strategies on fiber to construct novel capsid-modified oncolytic adenoviral vectors on the basis of the hexon-chimaeric Ad5 vectors (engineered to circumvent anti-Ad5 immunity and liver tropism) constructed by our group previously. We hope that the new vectors will efficiently across the BBB and target to malignant glioma cells after intravenous injection. We aim to obtain new oncolytic adenoviral gene therapy vectors which are dual brain targeted and with strong anti-glioma activity, and provide a basis for malignant glioma treatment and the further clinical application of adenoviral vectors.

恶性胶质瘤是发病率最高的颅内肿瘤形式，其复发率和死亡率极高，目前临床上尚缺乏有效的治疗手段。以溶瘤腺病毒为载体的基因治疗是一种十分具有应用前景的恶性胶质瘤治疗新策略。然而目前临床研究中主要通过颅（瘤）内给药治疗胶质瘤，受病毒扩散能力的限制其效果并不理想且这种给药方式会给患者带来一定的安全风险和痛苦。通过静脉全身靶向给药是最佳的治疗途径，其中最为关键的问题是溶瘤腺病毒如何突破血脑屏障并靶向肿瘤细胞。本项目在课题组前期构建的具有可逃避预存免疫、降低肝富集等性质的适合全身给药的六邻体嵌合型腺病毒载体的基础上，设计利用全新的策略对纤维蛋白进行基因工程修饰，以使溶瘤腺病毒在通过静脉给药后能够高效地穿透血脑屏障并特异性靶向胶质瘤细胞，希望构建获得具有双级脑靶向性及更强抗恶性胶质瘤活性的新型溶瘤腺病毒基因治疗载体，为恶性胶质瘤基因治疗新策略的研究和溶瘤腺病毒载体的进一步人体临床应用提供基础。

恶性胶质瘤是发病率最高的颅内肿瘤形式，其复发率和死亡率极高，目前临床上尚缺乏有效的治疗手段。以溶瘤腺病毒为载体的基因治疗是一种十分具有应用前景的恶性胶质瘤治疗新策略。由于血脑屏障的存在及病毒自身感染性质的限制，包括溶瘤腺病毒在内的、进入临床研究阶段的溶瘤病毒对恶性胶质瘤治疗的给药方式均为瘤内（颅内）给药。这种给药方式病毒的扩散受限，不能有效感染距离给药位置较远的肿瘤细胞。因此，对于极易多中心发生、侵袭性强、易复发转移的恶性胶质瘤而言，瘤内给药的方式治疗效果有限。通过静脉全身靶向给药是最佳的治疗途径，其中最为关键的问题是溶瘤腺病毒如何突破血脑屏障并靶向肿瘤细胞。本项目在课题组前期构建的具有可逃避预存免疫、降低肝富集等性质的适合全身给药的六邻体嵌合型腺病毒载体的基础上，首先设计构建了一种纤维蛋白嵌合型溶瘤腺病毒CRAd5/K37，体内外的研究表明嵌合病毒能够显著提升溶瘤腺病毒对胶质瘤细胞的感染和杀伤能力。随后，又分别通过不同的修饰策略对CRAd5/K37进行了进一步优化，以进一步提升其穿透血脑屏障和靶向胶质瘤细胞的能力。结果表明，由于靶向肽的插入影响了纤维蛋白的表达，设计构建的一系列双级脑靶向肽修饰型溶瘤腺病毒载体均无法包装出病毒。经过策略优化，结合TRAIL表面修饰策略及纤维蛋白嵌合策略，最终成功构建了一个衣壳双修饰型靶向溶瘤腺病毒A4K37。结果显示，双修饰溶瘤腺病毒可以显著地增强CRAd5/K37对胶质瘤细胞的感染和杀伤，且能够通过转胞吞的方式穿透体外血脑屏障模型，但在小鼠脑原位肿瘤模型中未能实现脑靶向抑制胶质瘤。本项目的研究为溶瘤腺病毒治疗恶性胶质瘤提供了新的策略和思路，同时也为进一步优化并最终获得通过静脉给药后能够高效地穿透血脑屏障并特异性靶向胶质瘤细胞的新型溶瘤腺病毒基因治疗载体提供了基础和依据。