Targeting of siRNAs, genes and drugs to cancer cells

描述(由申请人提供)：该项目旨在识别可用于靶向针对肝肿瘤细胞的药物(小分子、生物制品、DNA、siRNA)的多肽。我们将使用噬菌体展示选择技术来鉴定这类多肽。无论是原发性肝癌还是转移性肝癌，其发病率都在增加。常规治疗并不十分成功，据报道，5年存活率在20%-35%之间。因此，非常需要通过靶向肝癌细胞的药物来改进治疗方法，提高治疗指数。RNA干扰正在为治疗癌症提供一种新的方法。人们普遍认为，有效传递siRNA或siRNA表达载体是实现这一目标的主要障碍。最近的工作集中在合成递送载体上，其中靶向性通过包含配体来传达。我们的工作表明，使用合成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多肽。然而，由于技术障碍，到目前为止，肝脏和肝脏肿瘤还没有通过噬菌体展示来探测。之前的研究使用了丝状噬菌体，这种噬菌体不能探测血管外细胞。本申请建议使用T7噬菌体，它很小，静脉注射后可以穿过血管毛孔。T7噬菌体展示载体被修改以防止被肝细胞非特异性摄取(p17突变)或被天然抗体识别(在截短的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。这些研究旨在为利用改良的T7噬菌体系统在体内筛选肝肿瘤靶向性多肽提供概念验证。在目标1中，将产生稳定表达绿色荧光蛋白(GFP)的CT26细胞。将开发出一种具有与亲本CT26相似的生长特征的品系，该品系在注入回结肠静脉时几乎形成独有的肝肿瘤。将产生几个T7噬菌体展示文库，扩增并注射到CT26-GFP肝癌小鼠体内。然后从肝脏中分离肿瘤细胞，回收噬菌体，扩增并重新注射，以进行额外的选择回合。经过几轮选择(4-6轮)后，将对单个噬菌体克隆的展示肽进行分析。将在转移较小和肿瘤较大的小鼠身上进行选择。小的转移需要细胞分选来分离CT26-GFP细胞。较大的肿瘤将通过解剖分离出来。在目标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。在目标3中，将更详细地研究已鉴定的多肽序列。将对多肽进行比对以识别基序，并与已知序列进行比较。将通过生物分布研究测试单个克隆的靶向性。原发性和转移性肝癌的发病率正在稳步上升。这种疾病很难治疗，5年存活率只有20%-35%。因此，非常需要改进治疗方法。这项拟议的研究将确定能够将药物(小分子、生物制品、DNA、siRNA)特异性地靶向于肝癌细胞的配体，从而显著提高治疗指数。

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.