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与其所在的正常宿主组织之间的差异，以及在开发这种理解的过程中开发特异性运输至体内肿瘤的试剂。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，在目前的治疗方法下，预期5年生存预后低于10%。本研究的具体目的是：1.通过一种新的体内选择方法，创造出特异性结合免疫缺陷小鼠体内人肝内结直肠癌的RNA结合基序，并具有在全身给药后体内运输至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适体是否对其蛋白靶点具有抑制作用和/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）在肝切除术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适体，其中携带肝结直肠转移的小鼠将通过尾静脉注射RNA寡核苷酸的随机文库。在短暂的循环后，收获肿瘤并取回RNA，逆转录，扩增并转录回RNA用于重复注射。重复该循环，直到RNA结合基序的群体被大量富集，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特异性结合的RNA适体。该过程将利用从多名患者收获的异种移植物进行，以努力检索与广谱患者相关的适体。还将进行选择，在交替的几轮中使用不同的异种移植。由此产生的RNA结合基序将探讨其交通到肝内肿瘤部位的能力。通过配体介导的方法，这些适体的靶标将被分离和测序。将测定RNA适体抑制其靶蛋白的功能以及体外和体内肿瘤增殖的能力。此外，将测定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。将通过切除肿瘤的cDNA阵列和IHC以及通过在携带异种移植物的小鼠中的体内运输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：拟议研究的目的是更好地定义人类肿瘤（转移性结直肠癌）和正常组织之间的差异。结直肠癌是美国癌症死亡的主要原因之一，当检测到扩散（转移）时很少能治愈。确定癌症和正常组织之间的差异将允许进一步开发能够抑制转移性结直肠癌同时使对正常组织的毒性最小化的靶向治疗剂。

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.