Pre-mRNA Trans-Splicing for Molecular Imaging of Cancer

需要新技术来促进基因表达的分析、定量和成像，以利用正在产生的丰富信息，例如，人类基因组和癌症基因组解剖项目。提出了剪接体介导的RNA反式剪接（SmaRT）的创新应用，以开发用于细胞、动物和潜在人类基因表达实时成像的新试剂。我们的实验室已经生产了不同的rna，称为ptm（预反式剪接分子）。这些PTM能够指导PTM与靶向前信使RNA之间的反式剪接反应。SmaRT反应的产物是一种新的嵌合mRNA，它可以编码几乎任何期望的基因产物，这些基因产物可以直接成像，也可以激活或捕获第二个报告分子。SMaRT反应的产物包含一个或多个目标内源性pre-mRNA的外显子和PTM传递的外显子或cDNA序列。我们建议研究针对癌症特异性或癌症相关基因：人乳头瘤病毒，人绒毛膜促性腺激素和EGF受体的PTMs编码标记荧光素酶外显子。本申请建议与加州大学洛杉矶分校医学中心的成像小组合作，为更先进的成像技术和设备提供指导。我们的具体目标包括证明SMART可以在pre-mRNA水平上用荧光素酶标记靶向临床相关基因。后续研究将通过分子文库提高PTMs的效率和特异性。实时分子成像。剪接体介导的RNA反式剪接实时分子成像将极大地促进癌症基因单拷贝PCR检测的临床前动物研究。它也可以成像的组织分布的转基因和载体，一个有吸引力的替代单拷贝PCR检测。在小动物癌症模型中也有可能识别转移。如果成功的话，这些动物研究可能会导致人类实时诊断癌症特异性RNA谱。SMaRT是一种有吸引力的单一靶向RNA，具有实时分析基因表达的潜力。

New technologies are needed to facilitate the analysis, quantitation and imaging of gene expression to exploit the wealth of information being generated, for example, by the Human Genome and Cancer Genome Anatomy Procect. The innovative use of Spliceosome Mediated RNA Trans-splicing (SmaRT) to develop new agents for real time imaging of gene expression within cells, animals, and potentially humans is proposed. Our laboratory has produced different RNAs, known as PTMs (Pre-Trans-splicing Molecules). These PTMs are capable of directing trans-splicing reactions between the PTM and a targeted pre-messenger RNA. The product of a SmaRT reaction is a novel chimeric mRNA, which can encode virtually any desired gene product that may be imaged directly or that can activated or capture a second reporter molecule. The product of a SMaRT reaction contains one or more exons of the target endogenous pre-mRNA and a exonic or cDNA sequence delivered by the PTM. We propose studies to target cancer specific or cancer associated genes: human papillomavirus, human chorionic gonadotropin and EGF receptor with PTMs encoding marker luciferase exons. This application proposes to define the collaboration with the imaging group at the UCLA Medical Center to provide guidance on more advanced imaging techniques and equipment. Our specific aims include the demonstration that SMART can target clinically relevant genes with the luciferase marker at the pre-mRNA level. Subsequent studies will increase the efficiency and specificity of the PTMs through a molecular library. Real time molecular imaging. Real time molecular imaging by spliceosome mediated RNA trans-splicing will greatly facilitate pre-clinical animal studies in cancer gene single copy PCR detection. It may also imaging of tissue distribution of transgenes and vectors, an attractive alternative to single copy PCR detection. It may also be possible to identify metastases in small animal cancer models. If successful these animal studies could lead to human diagnostics of real time cancer specific RNA profiles. SMaRT is attractive single it targets RNA and holds the potential for real time analysis of gene expression.