A hybrid microvesicle/virus vector for targeted gene transfer to the brain

描述（由申请人提供）：胶质母细胞瘤（GBM）的手术切除未能完全清除浸润性肿瘤组织，导致肿瘤复发。将抗肿瘤基因的病毒载体递送到剩余的恶性细胞具有防止这种复发的潜力。病毒载体的一个持久问题是直接注射后整个肿瘤中的转基因递送效率低下。由于脑和脑肿瘤都是高度血管化的， 通过脉管系统是广泛的转基因递送的有吸引力的替代方法。然而，当前的向量系统对于血管内（i. v.）由于肝脏摄取、血脑屏障（BBB）的不可穿透性和对病毒的预免疫性（例如，针对病毒衣壳的中和抗体），基因递送到大脑。因此，我们寻求可用于两个目的的替代策略：（1）允许腺相关病毒（AAV）载体特异性靶向脑肿瘤环境（BTE）和（2）屏蔽AAV衣壳免受中和抗体的影响。微泡（MV）是天然分泌的膜包围结构，已知其将蛋白质、RNA、miRNA和DNA递送至邻近细胞。越来越多的研究表明，它们可能适用于靶向基因/蛋白质转移。我们已经发现，在载体生产过程中，从培养基中分离的一部分AAV与MV相关。通过透射电子显微镜检查，AAV衣壳主要位于MV内部。我们已经发现，与标准细胞裂解物纯化的AAV载体相比，MV相关的AAV（称为MV-AAV）在向培养细胞的基因递送方面更有效。我们还表明，用磁性纳米颗粒表面标记MV-AAV允许通过使用放置在细胞培养孔底部的磁体进行定向AAV介导的基因转移。此外，最近的一项研究表明，在小鼠中静脉注射后，负载有siRNA的MV可以特异性靶向脑。该提议将测试MV-AAV在静脉内注射后可以靶向脑肿瘤环境以治疗性治疗肿瘤的假设。这项工作有可能提供一种非侵入性的方法，将强大的全球基因递送到大脑。 公共卫生相关性：在这项提案中，我们将设计靶向微泡相关AAV（MV-AAV）基因递送载体，用于小鼠多形性胶质母细胞瘤（GBM）脑肿瘤的基因治疗。通过在MV表面上表达已知穿过血脑屏障（BBB）的配体，我们预期AAV载体优先将基因递送至脑肿瘤环境。我们将首先使用AAV载体编码的萤火虫荧光素酶表达的生物发光成像来分析脑靶向效率。接下来，使用最有效的靶向方法，我们将通过使用组合药物/AAV编码的抗肿瘤蛋白治疗来进行抗GBM基因治疗。这项研究对癌症和神经退行性疾病具有广泛的意义，这些疾病需要对大脑进行全面的基因治疗。

DESCRIPTION (provided by applicant): Surgical resection of glioblastomas (GBM) fails to completely remove invasive tumor tissue, resulting in tumor recurrence. Virus vector delivery of anti-tumor genes to remaining malignant cells has potential to prevent this recurrence. An enduring issue with virus vectors is inefficient transgene delivery throughout the tumor after direct injection. As the brain and brain tumors are both highly vascularized, delivery of therapies via the vasculature is an attractive alternative approach for widespread transgene delivery. However, current vector systems are suboptimal for intravascular (i.v.) gene delivery to the brain due to liver uptake, the impenetrability of the blood-brain barrier (BBB), and pre-exisiting immunity to the virus (e.g. neutralizing antibodies to the virus capsid). We therefore sought alternative strategies which would serve two purposes: (1) to allow specific targeting of adeno-associated virus (AAV) vector to the brain tumor environment (BTE) and (2) shield AAV capsids from neutralizing antibodies. Microvesicles (MV) are naturally secreted, membrane-encompassed structures known to deliver proteins, RNA, miRNA, and DNA to neighboring cells. Accumulating research suggests they may be suitable for targeted gene/protein transfer. We have discovered that during vector production, a portion of AAV isolated from the media is associated with MV. AAV capsids were localized primarily on the inside of MV by transmission electron microscopic examination. We have found that the MV-associated AAV, termed MV-AAV, are more efficient at gene delivery to cultured cells compared to standard cell lysate purified AAV vectors. We have also shown that surface tagging MV-AAV with magnetic nanoparticles allows for directional AAV-mediated gene transfer via the use of a magnet placed on the bottom of the cell culture well. Furthermore a recent study showed that MV's loaded with siRNA could be specifically targeted to the brain after i.v. injection in mice. This proposal will test the hypothesis that MV-AAV can be targeted to the brain tumor environment after i.v. injection to therapeutically treat the tumor. This work has potential to provide a non-invasive means of robust and global gene delivery to the brain. PUBLIC HEALTH RELEVANCE: In this proposal we will engineer targeted microvesicle-associated AAV (MV-AAV) gene delivery vehicles for gene therapy against glioblastoma multiforme (GBM) brain tumors in mice. By expressing ligands known to cross the blood-brain barrier (BBB) on the MV surface we anticipate the AAV vector to preferentially deliver genes to the brain tumor environment. We will initially analyze brain targeting efficiency using bioluminescence imaging of AAV vector-encoded firefly luciferase expression. Next using the most efficient targeting approach, we will perform anti-GBM gene therapy by using a combined drug/AAV encoded anti-tumor protein therapy. This study has broad implications for cancer and neurodegenerative diseases where global gene therapy to the brain is desired.