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

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.

项目概要/摘要常染色体显性多囊肾病 (ADPKD) 影响着全球超过 1200 万人。治疗仅稍微延迟肾脏和肝脏中充满液体的囊肿的持续生长导致肾衰竭，在某些情况下会导致严重的疼痛和腹胀。 ADPKD 患者的原发病基因 PKD1/Polycystin-1(PC1) 存在非截短突变。其中重要的子集编码的 PC1 版本无法成熟到细胞表面的作用位点但可能是至少部分有功能的 PC1 蛋白“剂量”——PC1 蛋白的功能量。然而，其作用位点与疾病严重程度相关，增加 PC1 剂量的可行方法。之前尚未被确定用于治疗评估。我们为编码蛋白质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) 基因突变的患者。也有因PC1剂量不足而得肾囊肿、肝囊肿的人，很多都急需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 PC1 的产量 Pkd1 对囊肿形成具有显着的拯救作用，我们追踪增加 PC1 蛋白表达。在这些 ER 基因突变的患者和大部分 PKD1 非突变患者中截短突变将显着减少囊肿负担。根据支持性的初步数据，我们已经确定人类 PKD1 的 5' 非翻译区包含可能高度相关的上游开放阅读框（uORF）翻译会分散核糖体的注意力。我们的数据表明，阻断 PKD1 uORF 翻译会产生许多- 作为使用反义的临床疗法，可以实现 PC1 翻译的倍数增加。寡核苷酸（ASO）是批准用于其他疾病的疗法。表征废除人 PKD1 uORF 翻译以增加 PC1 表达的效果，生成体内模型，用于对这种治疗对囊性疾病严重程度的临床前评估。第一个目标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 PKD1 5’UTR 来测试 uORF 翻译对 PC1 表达的影响，以及 (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果对于目标 2，我们将使用 CRISPR 使表位标记的 Pkd1-V5 小鼠的 5'UTR 人性化。有或没有 uORF 起始密码子编辑，我们将测试废除的 uORF 的临床益处。在我们的 PC1 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评估临床前治疗的模型。