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定义肝结直肠癌转移瘤与它们所在的正常宿主组织之间的差异，并在发展这种理解的过程中开发特异性传输到体内肿瘤的试剂。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期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创建RNA结合基序，特异性结合居住在免疫缺陷小鼠体内的人类肝内结直肠癌，并且在体内系统给药后具有传输到肿瘤沉积部位的能力。2. 确定这些肿瘤特异性RNA结合基序的蛋白靶点，并确定它们是否具有与RNA适体一致的结合特征。3.确定所鉴定的RNA适体是否对其靶蛋白具有抑制作用和/或它们是否能够护送治疗部分到肝内肿瘤。4. 确定这些相同的靶点是否也存在于申请人（BC）在肝切除术时收获的更广泛的人类结肠直肠癌转移灶中。为了实现这些目标，RNA适体将通过一种新的选择策略产生，即通过尾静脉向患有肝结肠直肠癌的小鼠注射随机的RNA寡核苷酸库。经过一段短暂的循环后，采集肿瘤，提取RNA，进行逆转录、扩增，并转录回RNA进行重复注射。这个循环重复，直到RNA结合基序的数量大量富集，此时进行克隆和测序。通过这种机制产生的RNA适体将进行体外结合试验，以鉴定那些与肿瘤组织具有特异性结合的RNA适体。该过程将利用来自多个患者的异种移植来检索与广泛患者相关的适配体。选择也将在交替的轮次中使用不同的异种移植。由此产生的RNA结合基序将探索其运输到肝内肿瘤部位的能力。通过配体介导的方法，这些适体的目标将被分离和测序。RNA适体抑制其靶蛋白功能和体外和体内肿瘤增殖的能力将被确定。此外，RNA适体：细菌毒素缀合物在体外和体内影响细胞毒性的能力将被确定。将通过cDNA阵列和切除肿瘤的免疫组化以及携带异种移植的小鼠体内转运研究来探索适配体与广谱mCRC患者的相关性。

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.