Targeting of siRNAs, genes and drugs to cancer cells

描述（由申请人提供）：该项目旨在鉴定可用于靶向药物（小分子，生物制剂，DNA, siRNA）的肽，特别是针对肝脏肿瘤细胞。我们将使用噬菌体展示选择技术来鉴定这些肽。肝癌的发病率，无论是原发性还是转移性，都在增加。常规治疗并不十分成功，据报道5年生存率在20-35%之间。因此，迫切需要通过药物特异性靶向肝癌细胞，改进治疗方法，提高治疗指标。RNA干扰为治疗癌症提供了一种新的手段。人们普遍认为，siRNA或siRNA表达载体的有效递送是实施的主要障碍。最近的工作集中在合成递送载体上，其中靶向特异性是通过配体的包涵来传递的。我们的研究表明，利用合成的递送载体可以有效地将siRNA递送到肝细胞。通过噬菌体展示选择技术已经鉴定出许多靶向肽。然而，由于技术上的障碍，到目前为止，噬菌体展示尚未对肝脏和肝脏肿瘤进行探测。以前的研究使用的是丝状噬菌体，它不能探测血管外细胞。本应用建议使用T7噬菌体，其体积小，静脉给药后可通过血管孔。对T7噬菌体展示载体进行修饰，以防止肝细胞（p17突变）的非特异性摄取或天然抗体的识别（在截断的p10B上显示低拷贝数）。非特异性噬菌体摄取以前阻碍了对肝脏或肝脏肿瘤的有效探测。这些研究旨在为使用改良的T7噬菌体系统在体内选择肝脏肿瘤靶向肽提供概念证明。在Aim 1中，将生成一个稳定表达绿色荧光蛋白（GFP）的CT26细胞。将开发一个具有与母体CT26相似生长特征的细胞系，将其注射到回结肠静脉后几乎完全形成肝脏肿瘤。几个T7噬菌体展示文库将被生成、扩增并注射到CT26-GFP肝肿瘤小鼠体内。然后将肿瘤细胞从肝脏中分离出来，回收噬菌体，扩增并重新注射以进行额外的选择轮。经过几轮筛选（4-6轮），对单个噬菌体克隆显示的肽进行分析。选择将在小转移和较大肿瘤的小鼠中进行。小的转移需要细胞分选分离CT26- GFP细胞。较大的肿瘤将通过解剖分离。在Aim 2中，选择研究将在人类癌细胞系（HT-29，仅切除较大的肿瘤）中进行。在Aim 3中，将更详细地研究鉴定的肽序列。多肽将被对齐以识别基序并与已知序列进行比较。个体克隆将通过生物分布研究测试靶向特异性。原发性和转移性肝癌的发病率正在稳步上升。该病难以治疗，5年生存率仅为20-35%。因此，我们非常需要改进治疗方法。拟议的研究将确定配体，使药物（小分子，生物制剂，DNA, siRNA）特异性靶向肝癌细胞，从而显着提高治疗指数。

DESCRIPTION (provided by applicant): This project aims to identify peptides that can be used to target drugs (small molecules, biologicals, DNA, siRNA) specifically to liver tumor cells. We will use phage display selection techniques to identify such peptides. The incidence of liver cancer, either primary or metastatic, is increasing. Conventional treatments have not been very successful, with reported 5-year survival rates between 20-35%. Thus, there is a great need for improved treatments with better therapeutic indices, by targeting drugs specifically to liver cancer cells. RNA interference is providing a novel means for treating cancer. It is widely recognized that effective delivery of siRNA or siRNA expression vectors is the main hurdle for implementation. Recent work has focused on synthetic delivery vehicles, in which targeting specificity is conveyed by the inclusion of ligands. Our work has shown effective delivery of siRNA to hepatocytes using synthetic delivery vehicles. Many targeting peptides have been identified through the phage display selection technique. However, the liver and liver tumors have so far not been probed by phage display because of technical hurdles. Previous studies have used filamentous phage, which cannot probe extravascular cells. This application proposes to use T7 phage, which are small and can pass through vessel pores after intravenous delivery. The T7 phage display vector is modified to prevent non-specific uptake by liver cells (p17 mutation) or recognition by natural antibodies (low copy number display on truncated p10B). Non-specific phage uptake has previously prevented effective probing of the liver or liver tumors. These studies are designed to provide proof-of-concept for using the modified T7 phage system for selecting liver tumor-targeting peptides in vivo. In Aim 1, a CT26 cell stably expressing green fluorescent protein (GFP) will be generated. A line will be developed with similar growth characteristics as the parent CT26, which forms near-exclusively liver tumors upon injection into the ileocolic vein. Several T7 phage display libraries will be generated, amplified and injected into mice with CT26-GFP liver tumors. Tumor cells will then be isolated from the liver, phage recovered, amplified and re-injected for additional selection rounds. After several selection rounds (4-6), the displayed peptide of individual phage clones will be analyzed. Selections will be performed in mice with small metastasis and larger tumors. The small metastases will require cell sorting to isolate CT26- GFP cells. The larger tumors will be isolated by dissection. In Aim 2, the selection studies will be performed with a human cancer cell line (HT-29, excision of larger tumors only). In Aim 3, the identified peptide sequences will be studied in more detail. Peptides will be aligned for identification of motifs and compared to known sequences. Individual clones will be tested for targeting specificity by biodistribution studies.The incidence of primary and metastatic liver cancer is steadily increasing. The disease is difficult to treat with 5-year survival rates of only 20-35%. Thus, there is a great need for improved therapies. The proposed research will identify ligands that will allow targeting of drugs (small molecules, biologicals, DNA, siRNA) specifically to liver cancer cells, thereby significantly improving the therapeutic index.