Redirection of Fas splicing as a cancer therapy

描述(由申请人提供)：这项建议的目标是开发特定的基于RNA的Lassos来重定向医学上重要的受体的剪接，并在细胞培养和动物模型中展示有效性。RNA Lassos是一种新颖的、专有的反义分子，它不是切割互补的RNA靶标，而是与其形成拓扑链，从而阻止基因表达。重定向剪接的能力可以使人们将任何与膜结合的受体转化为可溶的诱饵。为了建立这一方法的原则证明，我们选择了Fas，它是细胞凋亡中的一个关键受体，已被广泛研究。已知Fas前mRNA是选择性剪接的。其中一个主要的剪接变异体跳过了外显子6，导致了一个无法转导杀伤信号的可溶性受体，并具有通过中和Fas配体保护邻近细胞免受死亡的额外好处。我们建议将RNA Lassos靶向内含子5/外显子6的剪接连接区，以导致外显子6的跳跃。我们将通过RT-PCR和免疫检测来测试淋巴细胞组织培养模型中剪接重定向的程度。Fas Lassos保护组织培养细胞免受Fas配体诱导的死亡的能力将通过细胞凋亡检测来评估。在第二阶段，我们将评估Fas Lassos在组织培养和小鼠模型中保护原代T细胞免受表达Fas配体的癌细胞的能力。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop specific RNA-based Lassos to redirect splicing of medically important receptors and demonstrate efficacy in cell culture and animal models. RNA Lassos are novel, proprietary antisense molecules that, instead of cleaving a complementary RNA target, form a topological linkage with it and thereby block gene expression. The ability to redirect splicing could allow one to convert any membrane bound receptor into a soluble decoy. To establish proof-of-principle for this approach we have chosen Fas, a key receptor in apoptosis that has been extensively studied. The Fas pre-mRNA is known to be alternatively spliced. One of the main splice variants skips exon 6, resulting in a soluble receptor that cannot transduce the killing signal and has the added benefit of protecting neighboring cells from death by neutralizing Fas ligand. We propose to target RNA Lassos to the intron 5/exon 6 splice junction region to cause exon 6 skipping. We will test the extent of splicing redirection in a lymphocyte tissue culture model by RT-PCR and immuno-detection. The ability of Fas Lassos to protect tissue culture cells from Fas ligand-induced death will be evaluated by apoptosis assays. In Phase II, we will evaluate the ability of Fas Lassos to protect primary T cells from cancer cells expressing Fas ligand in tissue culture as well as a mouse model.