Profiling of Alternate mRNA Splicing in Cancer

描述（由申请人提供）：本提案的主要目的是证明5种选定基因（CD 44、CD 46、ErbB、Muc 1和VEGF）的交替剪接mRNA变体的高通量分析在结肠癌检测和分期中的可行性和适用性。最常见的分子分析方法是使用cDNA、PCR、组织和寡核苷酸阵列比较基因表达谱。人类基因组的完整序列已经揭示了大约35，000个基因，远远少于100，000个左右的蛋白质，这些蛋白质解释了人类生物学的复杂性。这种明显异常的一种解释是mRNA的选择性剪接。基因的可变剪接将对药物靶点和诊断生物标志物的发现和验证产生深远的影响。例如，在一个实施例中，迫切需要能够表征生物样品中所有mRNA变体的高通量技术。分析组织中mRNA剪接变体的相对变异提供了额外的分辨率水平，补充了传统的基因表达分析。这是一种创新的新方法，有望将基于基因的阵列的可扩展性与基于蛋白质组学的分子谱分析的灵敏度相结合。Jivan Biologics正在开发一种高度灵敏和可扩展的工艺，TransExpress TM，该工艺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、全长拼接和测量交替剪接形式基因的差异表达。该项目的主要人员将是首席研究员Subha Sinivasan博士和Jonathan Bingham，他们都在基因组学，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还将利用来自Manny战神博士（UCSC）和Doug Black博士（UCLA）的咨询服务，他们是mRNA交替剪接领域的杰出学术科学家。SBIR第一阶段的具体目标是：（1）确定优化正常和肿瘤结肠组织样本中Transexpress TMto mRNA剪接变体谱的条件，以及（2）通过优化寡核苷酸探针选择，获得正常和肿瘤结肠组织的共有mRNA变体谱。该可行性研究的成功将取决于获得相同基因的组成型表达寡核苷酸之间强度变化的显著降低，以及正常结肠组织与癌症结肠组织的谱的p值小于0.01。该项目采用的方法包括：（1）专有设计、合成和在载玻片上点样氨基标记的寡核苷酸，（2）这些微阵列与来自25个正常和50个癌症结肠组织样品的cy 3 - 1标记的cRNA杂交，以及（3）专有的计算分析，以拼接在一起，并确定mRNA的相对表达水平的交替剪接变异体在正常与结肠癌组织中这些技术将具有商业和学术效用，与美国国家癌症研究所（NCI）癌症诊断和治疗部癌症诊断计划（CDP）的研究兴趣一致。在成功完成该项目后，Jivan将使用类似的方法来研究更大的基因和变体，为不同阶段的结肠癌以及其他部位的癌症创建基于寡核苷酸的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.