Ligand-Directed Tumor Targeting in Preclinical Models

描述(申请人提供)：肿瘤血管内皮细胞表达在正常血管中检测不到的血管生成标记物。我们开发了一种体内选择系统，在该系统中，能够归巢到肿瘤血管生成的噬菌体可以在静脉给药后从噬菌体展示肽库中恢复。利用这一策略，我们已经分离出了几个肿瘤归巢噬菌体。其中包括展示双环RGD(RGD-4C)的噬菌体。我们已经证明，这种多肽与肿瘤血管生成系统中的αv整合素结合。基于我们用RGD-4C噬菌体靶向肿瘤的体内研究，我们认为RGD-4C-噬菌体适合于在体内生成靶向血管新生血管和肿瘤细胞的基因表达载体。我们假设，结合真核和原核病毒的良好生物学属性，可能会促进系统的基因治疗靶向应用。在这里，我们介绍了一个由腺相关病毒(AAV)和M13噬菌体的基因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的多肽。我们发现，与标准噬菌体载体相比，噬菌体/AAV嵌合体提供了更好的基因表达，并且AAV反向末端重复序列的加入可能保护嵌合体免受染色质诱导的基因沉默。我们的原型只需一次全身剂量就能以足够的效率传递自杀基因，从而导致血管破坏，从而显著抑制肿瘤生长。综上所述，我们的结果表明，噬菌体/AAV嵌合体可以有效地介导体内的基因传递。这类新的混合病毒可能被证明是更好的靶向基因治疗载体。我们的具体目标是：(I)开发高效的噬菌体载体，将基因靶向输送到血管新生血管和肿瘤细胞；(Ii)评估靶向输送促凋亡基因的基因表达和血管生成抑制的效率。将使用人类肿瘤异种移植模型，(Iii)检测靶向向肿瘤输送促凋亡基因与放射治疗相结合的治疗效果。将进行旨在评估抑制新生血管的治疗实验，以及通过靶向传递功能基因和辐射对肿瘤生长和生存的影响。相关性：本提案中概述的研究集中于具有选择性血管生成和肿瘤细胞靶向性的多肽序列的使用。我们将寻求验证探针作为基因靶向方法中的递送载体。载体靶向将代表着癌症管理方面的重大进步。

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.