In Vivo Selection of Tumor-Specific RNA Binding Motifs

描述(由申请人提供)：本申请的总体目标是更好地确定肝脏结直肠癌转移瘤与它们所在的正常宿主组织之间的差异，并在发展这一认识的过程中开发专门输送到体内肿瘤的试剂。认识和理解这些差异将有助于制定针对转移性结直肠癌患者的治疗策略，这些患者作为一个群体，使用目前的治疗方法预计5年生存率低于10%。本研究的具体目的是：1.通过一种新的体内选择过程来创建RNA结合基序，该基序能够特异性地结合免疫缺陷小鼠体内的人肝内结直肠癌，并具有全身给药后体内转运到肿瘤沉积部位的能力。2.确定这些肿瘤特异性RNA结合基序的蛋白质靶点，并确定它们是否具有与RNA适配子一致的结合特性。3.确定已鉴定的RNA适配子是否对其蛋白质靶标具有抑制作用和/或它们是否能够将治疗部分护送到肝内肿瘤。4.确定这些相同的靶点是否也存在于申请人(BC)在肝切除时采集的更广泛的人类结直肠转移瘤中。为了实现这些目标，将通过一种新的选择策略产生RNA适配子，即通过尾静脉向携带肝脏结直肠转移瘤的小鼠注射随机的RNA寡核苷酸文库。在短暂的循环后，肿瘤被收获，RNA被提取，反转录，扩增，并转录回RNA用于重复注射。重复这个循环，直到RNA结合基序的数量大量丰富，然后进行克隆和测序。然后，通过这种机制产生的RNA适配子将进行体外结合试验，以确定那些与肿瘤组织具有特异性结合的RNA适配子。这一过程将利用从多个患者身上收获的异种移植来进行，以努力回收与广泛患者相关的适体。还将进行选择，从而在交替轮次中使用不同的异种移植。由此产生的RNA结合基序将在它们运输到肝内肿瘤部位的能力方面进行探索。通过配体介导的方法，这些适配子的靶标将被分离和测序。将确定RNA适配子抑制其目标蛋白的功能以及体外和体内肿瘤增殖的能力。此外，还将确定RNA适配子：细菌毒素结合物在体外和体内影响细胞毒性的能力。将通过切除肿瘤的cDNA阵列和IHC以及通过携带异种移植的小鼠体内转运研究来探索适体与广泛的mCRC患者的相关性。 公共卫生相关性：这项拟议研究的目的是更好地确定人类肿瘤(转移性结直肠癌)和正常组织之间的差异。在美国，结直肠癌是癌症死亡的主要原因之一，当发现扩散(转移)时，很难治愈。明确癌症和正常组织之间的区别将允许进一步开发能够抑制转移性结直肠癌的靶向治疗药物，同时将对正常组织的毒性降至最低。

DESCRIPTION (provided by applicant): It is the overall goal of this application to better define the differences between hepatic colorectal cancer metastases and the normal host tissues within which they reside and in the process of developing this understanding to develop reagents that specifically traffic to in vivo tumors. Recognizing and understanding these differences will help to create therapeutic strategies that are targeted to patients with metastatic colorectal cancer who as a group face an expected 5-year survival prognosis of less than 10% with current therapies. The specific aims of this study are to: 1.To create RNA binding motifs through a novel in vivo selection process that specifically bind human intrahepatic colorectal cancers residing in immunodeficient mice and that possess the ability to traffic in vivo to the sites of tumor deposits upon systemic administration. 2. To define the protein targets of these tumor-specific RNA binding motifs and ascertain whether they possess binding characteristics consistent with RNA aptamers. 3.To determine if the identified RNA aptamers possess inhibitory actions on their protein target and/or whether they are capable of escorting therapeutic moieties to intrahepatic tumors. 4. Determine whether these same targets are also present in a broader spectrum of human colorectal metastases harvested by the applicant (BC) at the time of hepatic resection. To accomplish these aims, RNA aptamers will be generated through a novel selection strategy whereby mice bearing hepatic colorectal metastases will be injected via tail vein with a random library of RNA oligonucleotides. After a brief period of circulation, tumors are harvested and RNA retrieved, reverse transcribed, amplified, and transcribed back to RNA for repeat injection. This cycle is repeated until the population of RNA binding motifs is heavily enriched at which point cloning and sequencing is then performed. RNA aptamers created through this mechanism will then undergo in vitro binding assays to identify those with specific binding for tumor tissue. This process will be performed utilizing xenotransplants harvested from multiple patients in an effort to retrieve aptamers relevant to a broad spectrum of patients. Selections will also be carried out whereby different xenotransplants are utilized in alternate rounds. The resulting RNA binding motifs will be explored in their ability to traffic to the site of intrahepatic tumors. Through a ligand-mediated approach, the target of these aptamers will be isolated and sequenced. The ability of the RNA aptamers to inhibit the function of their target proteins and the proliferation of in vitro and in vivo tumors will be determined. In addition, the ability of RNA aptamer:bacterial toxin conjugates to effect cytotoxicity in vitro and in vivo will be determined. The relevance of the aptamers to a broad spectrum of patients with mCRC will be explored via cDNA arrays and IHC of resected tumors and through in vivo trafficking studies in mice bearing xenotransplants. PUBLIC HEALTH RELEVANCE: It is the purpose of the proposed research to better define the differences between human tumors (metastatic colorectal cancer) and normal tissue. Colorectal cancer is one of the leading causes of cancer death in the United States and rarely curable when spread (metastases) are detected. Defining the differences between cancer and normal tissues will allow for the further development of targeted therapeutic agents that are capable of inhibiting metastatic colorectal cancer while minimizing toxicity to normal tissues.