Profiling of Alternate mRNA Splicing in Cancer

描述(申请人提供)：这项建议的主要目标是证明高通量分析5个选定基因-CD44、CD46、ErbB、MUC1和VEGF-的交替剪接mRNA变体在结肠癌检测和分期中的可行性和适用性。分子图谱最常见的方法是使用cDNA、聚合酶链式反应、组织和寡核苷酸阵列比较基因表达图谱。人类基因组的完整序列揭示了大约3.5万个基因，远远少于解释人类生物学复杂性的大约10万个蛋白质。这种明显异常的一种解释是信使核糖核酸的交替剪接。基因的交替剪接将对发现和验证可用药靶点和诊断生物标记物产生深远影响，例如在癌症(NCI)中。迫切需要高通量技术来表征生物样本中的所有mRNA变体。分析组织中mRNA剪接变体的相对变异提供了一个额外的分辨率水平，补充了传统的基因表达分析。这是一种创新的新方法，它承诺将基于基因的阵列的可扩展性与基于蛋白质组学的分子图谱的敏感性相结合。Jivan Biologics正在开发一种高度敏感和可扩展的过程TransExpress TM，它独特地将微阵列技术与计算生物学和分子生物学技术相结合，用于发现、全长缝合和测量交替剪接形式的基因的差异表达。该项目的关键人员将是首席研究员苏布哈·西尼瓦桑博士和乔纳森·宾厄姆，他们两人都在基因组学、微阵列/基因芯片技术和生物信息学方面拥有丰富的行业经验。Jivan还将利用加州大学曼尼·阿雷斯博士(UCSC)和加州大学洛杉矶分校(UCLA)的道格·布莱克博士提供的咨询服务，他们是信使核糖核酸选择性剪接的杰出学术科学家。SBIR第一阶段的具体目标是：(1)确定优化转录TM的条件，以分析正常和肿瘤结肠组织样本中的mRNA剪接变体；(2)通过优化寡核苷酸探针选择，获得正常和肿瘤结肠组织一致的mRNA变体图谱。这项可行性研究的成功将取决于能否显著减少同一基因的组成性表达的寡核苷酸之间的强度差异，以及正常和癌症结肠组织的p值低于0.01。该项目中使用的方法包括：(1)专有设计、合成和在玻片上定位氨基标记的寡核苷酸；(2)将这些微阵列与来自25个正常和50个结肠癌组织样本的Cy3-1标记的cRNA杂交；(3)专有的计算分析，以缝合在一起并确定正常和结肠癌组织中mRNA选择性剪接变体的相对表达水平。这些技术将具有商业和学术用途，与国家癌症研究所(NCI)癌症诊断和治疗部癌症诊断计划(CDP)的研究兴趣一致。在这个项目成功完成后，Jivan将使用类似的方法处理更大的一组基因和变种，为结肠癌的不同阶段以及其他部位的癌症创建寡基mRNA签名。

DESCRIPTION (provided by applicant): The primary objective of this proposal is to demonstrate the feasibility and applicability of high-throughput profiling of alternate splice mRNA variants of five selected genes -CD44, CD46, ErbB, Muc1 and VEGF - in colon cancer detection and staging. The most common approach towards molecular profiling has been comparison of gene expression profiles using cDNA, PCR, tissue and oligonucleotide arrays. The complete sequence of the human genome has revealed approximately 35,000 genes, far less than the 100,000 or so proteins that account for the complexity of human biology. One explanation for this apparent anomaly is alternate splicing of mRNA. Alternate splicing of genes will have a profound impact in the discovery and validation of druggable targets and diagnostic biomarkers, e. g., in cancer (NCI).There is a dire need for high-throughput technologies capable of characterizing all mRNA variants within a biological sample. Profiling the relative variation in mRNA splice variants in tissues offers an additional level of resolution, complementing conventional gene expression analysis. This is an innovative new approach that promises to have the scalability of gene-based arrays combined with the sensitivity of proteomics-based molecular profiling. Jivan Biologics is developing a highly sensitive and scalable process, TransExpress TM that uniquely integrates microarray technology with computational biology and molecular biology techniques for discovery, full-length stitching and measurement of differential expression of alternately spliced forms of genes. The key personnel on this project will be Dr. Subha Sinivasan, Principal Investigator, and Jonathan Bingham, both of whom have extensive industry experience in genomics, microarray / gene chip technology and bioinfomatics. Jivan will also avail of consulting services from Dr. Manny Ares (UCSC) and Dr. Doug Black (UCLA), pre-eminent academic scientists in mRNA alternate splicing. The specific aims for SBIR Phase I are: (1) to determine the conditions to optimize Transexpress TMto profile mRNA splice variants in normal and neoplastic colon tissue samples, and (2) to obtain consensus mRNA variant profiles for normal and neoplastic colon tissues, by optimizing oligonucleotide probe selection. The success of this feasibility study will be depend on obtaining a significant reduction in the variation in intensities among constitutively expressed oligonucleotides for the same gene, and p-values of less than 0.01 for profiles from normal versus cancer colon tissues. The methodology used in this project comprises: (1) proprietary design, synthesis and spotting of aminelabeled oligonucleotides on glass slides, (2) hybridization of these microarrays with cy3-1abeled cRNA from 25 normal and 50 cancer colon tissue samples, and (3) proprietary computational analysis to stitch together and determine the relative expression levels of mRNA alternate splice variants in normal versus colon cancer tissues. These technologies will have both commercial and academic utility, consistent with the research interests of the Cancer Diagnosis Program (CDP) of the Division of Cancer Diagnosis and Treatment at the National Cancer Institutes (NCI). Upon successful completion of this project Jivan will use similar approach to a much larger set of genes and variants to create oligobased mRNA signatures for different stages of colon cancer as well as cancers in other sites.