Therapeutic rescue of Polycystin-1 protein expression by targeting PKD1 upstream open reading frames

项目摘要/摘要 常染色体显性遗传性多囊肾病(ADPKD)影响全球超过1200万人。可用 治疗只能轻微延缓进展中的肾脏和肝脏中充满液体的囊肿的持续生长。 导致肾功能衰竭，在某些情况下还会导致毁灭性的疼痛和腹胀。大约三分之一的人 ADPKD患者的原发病基因PKD1/多囊蛋白-1(PC1)存在非截断突变。一个 其中重要子集编码PC1的一个版本，该版本不能成熟到其在细胞表面的作用部位 然而，可能是一个至少部分功能的PC1蛋白。PC1“剂量”--PC1蛋白在 它的作用部位与疾病的严重程度相关。尽管如此，增加PC1剂量的可行方法 之前没有被确定为进行治疗评估。 我们对几个编码蛋白质的疾病基因的鉴定和特征做出了贡献。 PC1成熟所必需的内质网(ER)。这些基因发生突变的患者 由于PC1剂量不足，还会患上肾和肝囊肿，许多人迫切需要治疗。 通过增加小鼠的基因组拷贝数来增加PC1的产量 蛋白酪氨酸激酶1对囊性病变有明显的挽救作用。我们假设增加PC1蛋白的表达 在这些ER基因突变的患者中，以及在相当大一部分PKD1非 截断突变将极大地减轻囊性负担。 我们已经用支持的初步数据确定了人PKD15‘非翻译区包含 可能是高度相关的上游开放阅读框架(UORF)。UORF翻译使核糖体远离 翻译出预期的蛋白质。我们的数据表明，阻止PKD1uORF翻译将产生许多- PC1的翻译增加了一倍。阻断uORF翻译是一种使用反义基因的临床治疗方法 寡核苷酸(ASO)。ASO是被批准用于其他疾病的治疗方法。对于这项建议，我们将测试和 鉴定取消人PKD1uORF翻译对增加PC1表达和 建立体内模型，用于对囊性疾病严重程度的这种治疗进行临床前评估。为我们的 首先，我们将在体外评估两种独立的方法：(1)编辑uORF起始密码子序列 用CRISPR检测人PKD15‘端非编码区翻译对PC1表达的影响 优化ASOS表征空间位阻对uORF翻译和RNA二级结构的影响 在PC1表达上。对于目标2，我们将使用CRISPR来人源化我们的表位标记的PKD1-V5小鼠的5‘非编码区 带或不带uORF起始密码子编辑。我们将测试被废除的uORF的好处，以便在临床上 在PC1剂量依赖的小鼠模型中对囊性形成和严重性的有意义的影响和优化 用于评估临床前治疗的模型。

Project Summary/Abstract Autosomal dominant polycystic kidney disease (ADPKD) affects over 12 million people worldwide. Available therapies provide only a slight delay in ongoing growth of fluid-filled cysts in the kidney and liver that progress to kidney failure and in some cases devastating pain and abdominal distension. Approximately one third of ADPKD patients have non-truncating mutations in the primary disease gene PKD1/Polycystin-1(PC1). A significant subset of these encode a version of PC1 that fails to mature to its site of action at the cell surface yet may be an at least partially functional PC1 protein. PC1 “dosage”—the functional amount of PC1 protein at its site of action—correlates with disease severity. Nonetheless, feasible approaches to increase PC1 dosage had not previously been identified to evaluate therapeutically. We have contributed to the identification and characterization of several disease genes that encode proteins in the endoplasmic reticulum (ER) that are necessary for PC1 maturation. Patients with mutations in these genes also get kidney and liver cyst due to insufficient PC1 dosage, and many are in desperate need of treatmentsIn mouse models for these genes, increasing PC1 production by increasing a mouse’s genomic copy number of Pkd1 provides a striking rescue of cyst formation. We hypothesize that increasing PC1 protein expression in patients with mutations in these ER genes and in a substantial subset of patients with PKD1 non- truncating mutations will dramatically reduce cyst burden. We have identified, with supportive preliminary data, that the 5’ untranslated region of human PKD1 contains likely highly relevant upstream open reading frames (uORFs). uORF translation distracts ribosomes away from translating the intended protein. Our data suggests that blocking PKD1 uORF translation would produce a many- fold increase in translation of PC1. Blocking uORF translation is achievable as a clinical therapy using antisense oligonucleotides (ASO). ASOs are approved therapies for other diseases. For this proposal we will test and characterize the effect of abolishing human PKD1 uORF translation to increase PC1 expression and generate in vivo models for preclinical evaluation of this treatment on cystic disease severity. For our first aim we will evaluate two independent approaches in vitro: (1) edit uORF initiation codon sequence in the human PKD1 5’UTR using CRISPR to test the effect of uORF translation on PC1 expression, and (2) test and optimize ASOs to characterize the effect of steric inhibition of uORF translation and RNA secondary structure on PC1 expression. For aim 2 we will use CRISPR to humanize the 5’UTR of our epitope-tagged Pkd1-V5 mouse with or without uORF initiation codon edits. We will test the benefit of abolished uORFs to have a clinically meaningful effect on cyst formation and severity in our PC1 dosage-dependent mouse models and optimize models to evaluate for preclinical therapies.