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

描述(申请人提供)：手术切除胶质母细胞瘤(GBM)未能完全切除浸润性肿瘤组织，导致肿瘤复发。病毒载体将抗肿瘤基因传递到残留的恶性细胞中，有可能防止这种复发。病毒载体的一个长期问题是，在直接注射后，转基因在整个肿瘤中的传递效率低下。由于脑部和脑肿瘤都是高度血管化的，治疗的交付 通过血管系统是广泛转基因传递的一种有吸引力的替代方法。然而，目前的载体系统对于血管内(静脉)是次优的。由于肝脏摄取、血脑屏障(BBB)的不可穿透性以及对病毒的预先存在的免疫力(例如，针对病毒衣壳的中和抗体)，基因传递到大脑。因此，我们寻求可用于两个目的的替代策略：(1)使腺相关病毒(AAV)载体特异性靶向脑瘤环境(BTE)；(2)保护AAV衣壳免受中和抗体的攻击。微泡(MV)是一种自然分泌的膜包裹结构，可将蛋白质、RNA、miRNA和DNA运送到邻近细胞。越来越多的研究表明，它们可能适合进行有针对性的基因/蛋白质转移。我们发现，在载体生产过程中，从介质中分离出的一部分AAV与MV有关。通过透射电子显微镜观察，AAV衣壳主要定位于MV的内侧。我们发现，与标准的细胞裂解液纯化的AAV载体相比，MV相关的AAV，称为MV-AAV，在向培养细胞传递基因方面更有效。我们还展示了用磁性纳米颗粒标记MV-AAV的表面允许通过使用放置在细胞培养底部的磁铁来定向AAV介导的基因转移。此外，最近的一项研究表明，携带siRNA的MV在静脉注射后可以特异性地靶向大脑。给小鼠注射。这一提议将检验MV-AAV在静脉注射后可以靶向脑瘤环境的假设。用于治疗肿瘤的注射。这项工作有可能提供一种非侵入性的、强大的、全球的基因传递到大脑的方法。

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.