Investigating the Effects of Reducing Nonsense-Mediated mRNA Decay Efficiency

 描述(由申请人提供)：大约30%的疾病相关基因损伤在mRNA中产生过早终止密码子(PTC)。PTC通过以下方式减少基因表达：1)在合成全长多肽之前终止mRNA的翻译；2)引发无义介导的mRNA衰退(NMD)，这是一种mRNA监控途径，降解含有PTC的mRNAs以阻止其翻译。在携带PTC的患者中，NMD效率的差异改变了许多遗传病的遗传模式和临床严重程度1，2。此外，NMD限制了抑制框架内PTC翻译终止以恢复全长功能蛋白2-5的治疗的有效性。因此，NMD衰减是一种潜在的治疗方法，可以缓解PTCS引起的蛋白质缺乏。然而，NMD影响内源性基因的表达、多种细胞通路和发育6。此外，NMD因子缺乏与智能障碍有关7、8。这表明强烈的NMD抑制会产生有害的影响。 我们假设，在胚胎发育后，可以施加一定程度的NMD抑制，而不会产生有害后果。以前的NMD缺失小鼠不适合在体内检测NMD衰减，因为在胚胎发育过程中NMD失活会导致死亡9。为了在体内更严格地检测NMD抑制的后果，我们产生了新的NMD缺陷TG(DnUPF1)小鼠，它表达可诱导的显性负UPF1(DnUPF1)NMD因子。通过以可诱导的方式表达dnUPF1，我们可以：1)绕过NMD在发育过程中的抑制，以允许生存；2)在各种组织中以受控的方式减弱温和和强烈水平的NMD。我们将使用TG(DnUPF1)小鼠来检查胚胎发育后减缓NMD效率的后果。这只小鼠将使我们能够检查NMD的生物学，并确定NMD是否是一个可行的治疗靶点。具体目的1.检测体内抑制NMD对哺乳动物基因表达、形态、生理和行为的影响。我们将在TG(DnUPF1)小鼠中诱导dnUPF1的表达，在不同程度上抑制NMD。然后，我们将对TG(DnUPF1)小鼠进行全面的鉴定，以确定：1)NMD抑制能否在较长时间内保持；2)中等水平的NMD抑制是否能够实现而不产生有害影响；3)可控的、长期的中等水平的NMD抑制对哺乳动物的基因表达、形态、生理和行为的影响。具体目的2.检测NMD衰减对体内PTC抑制的影响。我们已经将dnUPF1转基因引入了一只携带基因组无义突变的小鼠，并代表了MPS I-H(Hurler综合征)10-13的模型。我们将研究dnUPF1的表达如何影响含有PTC的mRNA的丰度以及通过PTC抑制恢复的全长蛋白的丰度。

 DESCRIPTION (provided by applicant): ~30% of all disease-associated gene lesions generate a premature termination codon (PTC) within an mRNA 2. PTCs reduce gene expression by: 1) terminating translation of a mRNA prior to the synthesis of a full-length polypeptide; 2) eliciting nonsense-mediated mRNA decay (NMD), an mRNA surveillance pathway that degrades PTC-containing mRNAs to prevent their translation. Variability in NMD efficiency among patients that carry PTCs alters the inheritance pattern and the clinical severity of numerous genetic diseases 1, 2. In addition, NMD limits the effectiveness of therapies that suppress translation termination at in-frame PTCs to restore full-length, functional protein 2-5. NMD attenuation therefore represents a potential therapeutic approach to alleviate protein deficiencies caused by PTCs. However, NMD influences endogenous gene expression, multiple cellular pathways, and development 6. In addition, NMD factor insufficiency is associated with intellectual disabilities 7, 8. This indicates that strong NMD inhibition produces harmful effects. We hypothesize that a level of modest NMD inhibition after embryonic development can be imposed without deleterious consequences. Previous NMD-null mice were unsuitable to examine NMD attenuation in vivo because NMD inactivation during embryonic development results in lethality 9. To more rigorously examine the consequences of NMD inhibition in vivo, we generated novel NMD-deficient Tg(dnUPF1) mice that express an inducible dominant-negative UPF1 (dnUPF1) NMD factor. By expressing dnUPF1 in an inducible manner we can: 1) circumvent NMD inhibition during development to allow viability; 2) attenuate NMD at both mild and strong levels in a controlled manner in various tissues. We will use Tg(dnUPF1) mice to examine the consequences of moderating NMD efficiency after embryonic development. This mouse will enable us to examine NMD biology and determine whether NMD is a viable therapeutic target. Specific Aim 1. Examine the effect of in vivo NMD inhibition on mammalian gene expression, morphology, physiology, and behavior. We will induce dnUPF1 expression in a manner that inhibits NMD by various degrees in Tg(dnUPF1) mice. We will then conduct a comprehensive characterization of Tg(dnUPF1) mice to determine: 1) whether NMD inhibition can be maintained over extended periods; 2) whether a level of moderate NMD inhibition can be achieved without deleterious effects; 3) the effects of controlled, long-term moderate NMD inhibition on mammalian gene expression, morphology, physiology, and behavior. Specific Aim 2. Examine the effect of NMD attenuation on PTC suppression in vivo. We have introduced the dnUPF1 transgene into a mouse that carries a genomic nonsense mutation and represents a model for MPS I-H (Hurler syndrome) 10-13. We will examine how dnUPF1 expression affects the abundance of the PTC- containing mRNA as well as the abundance of full-length protein restored by PTC suppression.