Ligand-Directed Tumor Targeting in Preclinical Models

描述（由申请方提供）：肿瘤血管中的内皮细胞表达在正常血管中检测不到的血管生成标志物。我们已经开发了一种体内选择系统，其中能够归巢到肿瘤血管生成血管的噬菌体在静脉内施用后从噬菌体展示肽库中回收。使用这种策略，我们已经分离出几个肿瘤归巢噬菌体。其中包括展示双环RGD（RGD-4C）的噬菌体。我们已经表明，这种肽结合到α v整合素在肿瘤血管生成血管。基于我们用RGD-4C-噬菌体靶向肿瘤的体内研究，我们提出这样的肽适合于产生用于靶向递送基因到体内血管生成脉管系统和肿瘤细胞的载体。我们假设，结合真核和原核病毒的有利生物学属性可能有助于系统性基因治疗靶向应用。在这里，我们介绍了嵌合载体组成的基因元件从腺相关病毒（AAV）和M13噬菌体。这些噬菌体/AAV载体展示设计为靶向肿瘤脉管系统和肿瘤细胞的肽。我们表明，噬菌体/AAV嵌合体提供了改进的基因表达超过标准的噬菌体载体，并纳入AAV反向末端重复可能保护嵌合体从染色质诱导的基因沉默。我们的原型的单次全身剂量以足够的效率递送自杀基因以引起血管破坏，从而导致显著的肿瘤生长抑制。总之，我们的结果表明，噬菌体/AAV嵌合体可以有效地介导体内基因递送。这类新的杂交病毒可能被证明是上级靶向基因治疗载体。我们的具体目标是：（i）开发用于靶向递送基因至血管生成脉管系统和肿瘤细胞的有效的基于噬菌体的载体，（ii）评价靶向递送促凋亡基因时基因表达和血管生成抑制的效率。将使用人肿瘤异种移植物模型，（iii）检查将促凋亡基因靶向递送至肿瘤与放射疗法组合的治疗效果。将进行设计用于评价通过靶向递送功能基因和辐射抑制新血管形成、对肿瘤生长和存活的影响的治疗实验。相关性：本提案中概述的研究集中于使用具有选择性血管生成血管和肿瘤细胞靶向特性的肽序列。我们将寻求验证探针作为基因靶向方法中的递送载体。载体靶向将代表癌症管理的重大进展。

DESCRIPTION (provided by applicant): Endothelial cells in tumor vessels express angiogenic markers that are not detectable in normal vessels. We have developed an in vivo selection system in which phage capable of homing to tumor angiogenic vasculature are recovered from a phage display peptide library following intravenous administration. Using this strategy, we have isolated several tumor-homing phage. Among those were phage displaying a double cyclic RGD (RGD-4C). We have shown that this peptide bind to alpha v integrins in tumor angiogenic vasculature. Based on our in vivo studies targeting tumors with RGD-4C-phage, we propose that such peptide is suitable for generation of vectors for targeted delivery of genes to angiogenic vasculature and tumor cells in vivo. We hypothesized that combining the favorable biological attributes of eukaryotic and prokaryotic viruses may facilitate systemic gene therapy targeting applications. Here we introduce a chimeric vector consisting of genetic elements from adeno-associated virus (AAV) and M13 bacteriophage. These phage/AAV vectors display peptides designed to target the tumor vasculature and tumor cells. We show that the phage/AAV chimera provides improved gene expression over standard phage vectors and that incorporation of AAV inverted terminal repeats likely protects the chimera from chromatin-induced gene silencing. A single systemic dose of our prototype delivered a suicide gene at sufficient efficiency to cause vascular destruction that resulted in significant tumor growth suppression. Together, our results show that phage/AAV chimeras can effectively mediate gene delivery in vivo. This new class of hybrid viruses may prove superior targeted gene therapy vectors. Our Specific Aims are: (i) to develop efficient phage-based vectors for targeted delivery of genes to angiogenic vasculature and tumor cells, (ii) to evaluate the efficiency of gene expression and angiogenesis inhibition upon targeted delivery of proapoptotic genes. Human tumor xenograft models will be used, (iii) to examine the therapeutic effects of targeted delivery of proapoptotic genes to tumors in combination with radiation therapy. Therapy experiments designed to evaluate inhibition of neovascularization, effects on tumor growth and survival by targeted delivery of functional genes and radiation will be performed. Relevance: The studies outlined in this proposal focus on the use of peptide sequences with selective angiogenic vasculature- and tumor cell-targeting properties. We will seek to validate probes as delivery vehicles in gene targeting approaches. Vector targeting would represent a major advance in the management of cancer.