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

基本信息

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
项目类别：
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&apos 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

5&apos 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

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项目摘要

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.

项目摘要/摘要常染色体显性多囊性肾脏疾病（ADPKD）影响了全球超过1200万人。可用的疗法在肾脏和肝脏中充满液体的囊肿的持续生长仅略有延迟肾衰竭以及在某些情况下毁灭性疼痛和腹部张力。大约三分之一 ADPKD患者在原发性疾病基因PKD1/polycystin-1（PC1）中存在非截断突变。一个这些的重要子集编码一个未能成熟到其在细胞表面的动作部位的PC1版本但可能是至少部分功能性的PC1蛋白。 PC1“剂量” - PC1蛋白的功能量它的作用部位 - 与疾病的严重程度相关。但是，增加PC1剂量的可行方法以前尚未确定以治疗评估。我们为在编码蛋白质中的几种疾病基因的鉴定和表征做出了贡献 PC1成熟所必需的内质网（ER）。这些基因突变的患者由于PC1剂量不足，还可以获得肾脏和肝囊肿，许多人迫切需要治疗这些基因的鼠标模型，通过增加鼠标的基因组拷贝数来增加PC1的产生 PKD1提供了囊肿形成的打击救助。我们假设增加PC1蛋白表达在这些ER基因中突变的患者中，PKD1非 - 截断突变会大大减少囊肿燃烧。我们已经通过支持性的初步数据确定了人类PKD1的5'未翻译区域可能高度相关的上游开放式阅读框（UORFS）。 UORF翻译分散了核糖体的注意力翻译预期的蛋白质。我们的数据表明，阻止PKD1 UORF翻译会产生很多 PC1翻译的折叠增加。使用反义，阻止UORF翻译可以作为临床疗法来实现寡核苷酸（ASO）。 ASO是其他疾病批准的疗法。对于此建议，我们将测试和表征废除人类PKD1 UORF翻译以增加PC1表达和生成用于临床前评估囊性疾病严重程度的体内模型。为我们第一个目标我们将在体外评估两种独立的方法：（1）编辑UORF启动密码子序列人类PKD1 5'Utr使用CRISPR测试UORF翻译对PC1表达的影响，以及（2）测试和优化ASO以表征定位抑制UORF翻译和RNA二级结构的影响关于PC1表达。对于AIM 2，我们将使用CRISPR人性化我们表位标记的PKD1-V5鼠标的5'UTR 有或没有UORF启动密码子编辑。我们将测试废除UORF的好处，以拥有临床在我们的PC1剂量依赖鼠标模型中对囊肿形成和严重程度的有意义的影响并优化用于评估临床前疗法的模型。