Modulation of VEGF-A alternative splicing as new anti-angiogenic therapeutics

描述(申请人提供)：癌细胞的一个主要分子特征是基因与拮抗剪接异构体的异常剪接。其中一个基因是血管内皮生长因子-A(VEGF-A)，它可以剪接为标准的促血管生成异构体或反血管生成异构体。在大多数正常成人组织中，血管内皮生长因子-A主要作为抗血管生成b亚型被剪接，而促血管生成异构体在癌细胞中占主导地位，以促进新血管的生长。由于持续的血管生成对肿瘤的生长和转移至关重要，恢复肿瘤内正常的血管内皮生长因子-A的剪接为抗肿瘤提供了一种新的途径。长期目标和具体目标：我们建议通过操纵血管内皮生长因子-A剪接来开发一种新的抗血管生成的方法。这种方法将重新连接协调血管生成的自然剪接调控途径，从而抑制肿瘤生长。本项目有两个具体的目的：(1)特异性调控血管内皮生长因子-A剪接促进抗血管生成异构体；(2)确定血管内皮生长因子-A剪接调控作为抗血管生成治疗的体内疗效。研究设计与方法利用我们实验室最近开发的一项新技术，我们将产生新的工程剪接因子(ESFS)，将VEGF-A的剪接从血管生成亚型转变为抗血管生成亚型。“设计者”ESFS将在转移性乳腺癌和肺癌细胞中表达，并将在mRNA和蛋白质水平上测量抗血管生成的VEGF-A剪接异构体的诱导。作为补充，我们将使用反义寡核苷酸(ASO)将血管内皮生长因子-A剪接转移到抗原异构体。我们将进一步研究血管内皮生长因子-A下游信号通路对这种剪接调控的反应。此外，我们将使用各种基于细胞的分析方法来研究剪接调控是否导致血管生成的抑制，并测试剪接调控的非靶点效应。我们将进一步检测是否调节血管内皮生长因子-A剪接可以抑制肿瘤血管生成和生长，在异种移植的小鼠模型的人类癌症。我们将把ESFS或ASOS处理过的癌细胞注射到SCID小鼠体内，以确定剪接调节是否能抑制异种移植瘤的形成和生长。此外，我们将通过瘤内注射或系统地注射ESF或ASO来直接治疗已建立的异种移植瘤，并确定这种治疗是否可以抑制肿瘤的进展。我们还将把血管内皮生长因子-A的剪接调控与其他癌症治疗方法结合起来，看看它们在抑制活体动物肿瘤进展方面是否有协同作用。

DESCRIPTION (provided by applicant): A major molecular hallmark of cancer cells is the aberrant splicing of genes with antagonistic splice isoforms. One of such genes is the vascular endothelial growth factor-A (VEGF-A) that can be spliced as either canonical pro-angiogenic isoforms or anti-angiogenic isoforms. In most normal adult tissues, VEGF-A is predominantly spliced as the anti-angiogenic b isoforms, whereas the pro-angiogenic isoforms become dominant in cancer cells to promote the growth of new blood vessels. Since sustained angiogenesis is critical to tumor growth and metastasis, restoration of normal VEGF-A splicing of in tumors presents a new anti-tumor approach. Long-term objectives and specific aims: We propose to develop a novel anti-angiogenesis approach by manipulating VEGF-A splicing. Such method will rewire the natural splicing regulatory pathway that orchestrates angiogenesis, thus inhibiting tumor growth. There are two specific aims in this project: (1) Specifically modulate VEGF-A splicing to promote anti- angiogenic isoforms; (2) Determine the in vivo efficacy of VEGF-A splicing modulation as anti-angiogenic therapy. Research design and methods Using a new technique recently developed in our lab, we will generate novel engineered splicing factors (ESFs) to shift the splicing of VEGF-A from angiogenic isoforms to anti-angiogenic isoforms. The "designer" ESFs will be expressed in metastatic breast and lung cancer cells, and the induction of anti-angiogenic VEGF- A splicing isoforms will be measured at mRNA and protein levels. As a complementary approach, we will use antisense oligonucleotides (ASOs) to shift the VEGF-A splicing toward anti-antigenic isoforms. We will further examine the response of the VEGF-A downstream signaling pathway to such splicing modulation. In addition, we will investigate whether the splicing modulation leads to the inhibition of angiogenesis using a variety of cell-based assays, and test the off-target effects of splicing modulation. We will further examine if the modulation of VEGF-A splicing can inhibit the tumor angiogenesis and growth in xenograft mouse model of human cancers. We will inject cancer cells pretreated with ESFs or ASOs into SCID mice to determine if the splicing modulation can inhibit the xenograft tumor formation and growth. Further we will directly treat the established xenograft tumors with intratumoral injection or systematic delivery of ESF or ASOs, and determine if such treatment can inhibit tumor progression. We will also combine splicing modulation of VEGF-A with other cancer therapies to see if they have synergistic effect to inhibit tumor progression in live animals.