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

通过靶向 PKD1 上游开放阅读框来治疗性挽救 Polycystin-1 蛋白表达

• 批准号: 10575251
• 负责人: Whitney Elise Besse
• 金额: $ 12.56万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10575251
• 关键词: 5' Untranslated Regions; Affect; Alleles; Antisense Oligonucleotides; Autosomal Dominant Polycystic Kidney; Bacterial Artificial Chromosomes; Cell Line; Cell surface; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Cyst; Cystic kidney; Data; Disease; Endoplasmic Reticulum; Epithelium; Epitopes; FDA approved; Funding; Genes; Genetic Transcription; Genomics; Growth; Hepatic Cyst; Human; Human Cell Line; Impairment; In Vitro; Initiator Codon; Institution; Intervention; Kidney; Kidney Failure; Life; Liquid substance; Liver; Location; Luciferases; Messenger RNA; Modeling; Mus; Mutation; Open Reading Frames; PKD1 gene; PKD2 gene; PKD2 protein; PRKCSH protein; Pain; Pathogenicity; Patients; Peptides; Persons; Phenotype; Production; Proteins; Publishing; RNA; RNA Sequences; Renal tubule structure; Renilla Luciferases; Reporter; Ribosomes; Severities; Severity of illness; Site; Structure; Testing; Therapeutic; Therapeutic Intervention; Transfection; Translating; Translation Initiation; Translations; Western Blotting; Work; abdominal distension; base editing; design; dosage; effective therapy; functional loss; genome editing; genome-wide; in vivo; in vivo Model; interest; mouse model; mutant; novel strategies; patient subsets; polycystic kidney disease 1 protein; pre-clinical therapy; preclinical evaluation; preclinical study; protein expression; translational potential

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.

项目总结/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 治疗仅稍微延迟了肾脏和肝脏中充满液体的囊肿的持续生长 导致肾衰竭，有时还会出现致命的疼痛和腹胀。大约三分之一 ADPKD患者在原发疾病基因PKD 1/多囊蛋白-1（PC 1）中存在非截短突变。一 其中一个重要的亚群编码一种不能成熟到细胞表面作用位点的PC 1 还可以是至少部分功能性的PC 1蛋白。PC 1“剂量”-在200 mg/kg时PC 1蛋白的功能量。 其作用部位与疾病严重程度相关。然而，增加PC 1剂量的可行方法 以前没有被确定用于治疗评估。 我们已经对几种编码蛋白质的疾病基因的鉴定和表征做出了贡献， 内质网（ER）是PC 1成熟所必需的。这些基因发生突变的患者 由于PC 1剂量不足，也会出现肾囊肿和肝囊肿，许多人迫切需要治疗。 这些基因的小鼠模型，通过增加小鼠的基因组拷贝数来增加PC 1的产量， pkd 1提供了一个惊人的拯救囊肿形成。我们假设PC 1蛋白表达的增加 在这些ER基因突变的患者中，以及在PKD 1非 截短突变将显著降低囊肿负荷。 我们已经确定，与支持的初步数据，5'非翻译区的人PKD 1含有 可能高度相关的上游开放阅读框（uORF）。uORF翻译使核糖体远离 翻译目的蛋白质。我们的数据表明，阻断PKD 1 uORF翻译会产生许多- PC 1的翻译倍数增加。阻断uORF翻译作为使用反义核酸的临床疗法是可实现的。 寡核苷酸（阿索）。ASO被批准用于治疗其他疾病。对于这个建议，我们将测试和 表征消除人PKD 1 uORF翻译以增加PC 1表达的作用， 生成体内模型，用于临床前评估这种治疗对囊性疾病严重程度的影响。为我们 第一个目的，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， 使用CRISPR检测人PKD 1 5 'UTR，以测试uORF翻译对PC 1表达的影响，和（2）测试和 优化ASO以表征uORF翻译和RNA二级结构的空间抑制效应 PC 1表达。对于目标2，我们将使用CRISPR来人源化我们的表位标记的Pkd 1-V5小鼠的5 'UTR。 有或没有uORF起始密码子编辑。我们将测试废除的uORF的益处， 在我们的PC 1剂量依赖性小鼠模型中对囊肿形成和严重程度产生有意义的影响并优化 用于评估临床前治疗的模型。