Ligand-Directed Tumor Targeting in Preclinical Models

描述（由申请人提供）：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，在该系统中，噬菌体能够在静脉注射后从噬菌体展示肽库中恢复到肿瘤血管生成血管。使用这种策略，我们已经分离出几个肿瘤噬菌体。其中显示双循环RGD的噬菌体（RGD- 4c）。我们已经证明这种肽与肿瘤血管生成血管中的α v整合素结合。基于我们在体内用rgd - 4c噬菌体靶向肿瘤的研究，我们提出该肽适合生成载体，在体内将基因靶向递送到血管生成血管和肿瘤细胞。我们推测，结合真核病毒和原核病毒的有利生物学特性，可能有助于系统基因治疗靶向应用。本文介绍了一种由腺相关病毒（AAV）和M13噬菌体遗传元件组成的嵌合载体。这些噬菌体/AAV载体展示了针对肿瘤血管和肿瘤细胞设计的肽。我们发现噬菌体/AAV嵌合体在标准噬菌体载体上提供了更好的基因表达，并且AAV反向末端重复序列的结合可能保护嵌合体免受染色质诱导的基因沉默。我们的原型单次全身剂量就能以足够的效率传递自杀基因，导致血管破坏，从而显著抑制肿瘤生长。总之，我们的研究结果表明噬菌体/AAV嵌合体可以有效地介导基因在体内的传递。这类新的杂交病毒可能证明是优越的靶向基因治疗载体。我们的具体目标是：(i)开发高效的基于噬菌体的载体，用于将基因靶向递送到血管生成血管和肿瘤细胞中；（ii）评估靶向递送促凋亡基因时基因表达和血管生成抑制的效率。人类肿瘤异种移植模型将被使用，（iii）检查靶向传递促凋亡基因到肿瘤结合放射治疗的治疗效果。将进行治疗实验，以评估功能基因和放射靶向递送对新生血管的抑制作用，对肿瘤生长和生存的影响。相关性：本提案中概述的研究重点是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的运载工具。载体靶向将是癌症管理方面的一项重大进展。

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.