Discovering ADPKD Modifier Genes and Therapies via Zebrafish Genetics

ADPKD患者表现出高度可变的表型，其中遗传修饰物被认为起着重要作用。然而，由于缺乏有效的方法，遗传修饰物的身份在很大程度上仍然未知。在这种背景下，我们建议建立成年斑马鱼作为一种新的动物模型，使快速和系统地发现ADPKD的遗传修饰因子。我们已经为这项提案生成了大量的初步数据： 1)通过确定82个PKD相关基因中99%的同源性来证明斑马鱼模型的分子保守性；2)开发斑马鱼PKD1、PKD2和ift140的突变体，其中3个都是导致肾囊形成的已知ADPKD相关基因；3)论证了利用一种新的基于微同源介导的末端连接(MMEJ)的F0遗传学方法来筛选ADPKD修饰基因的可行性。这些初步数据促使我们提出了一个中心假设，即斑马鱼是发现ADPKD遗传修饰因子的保守和有效的活体脊椎动物模型。为了验证这一假设，我们提出了以下三个具体目标。在目标1中，我们将通过验证从基于MMEJ的Pilot F0基因筛查中出现的两个新的保护性ADPKD修饰剂来进一步证明我们策略的可行性。在目标2中，我们将探索使用斑马鱼模型来评估从人类患者群体中建议的ADPKD修饰物的可行性。在目标3中，我们将通过进一步开发测量斑马鱼肾脏体积和包囊的核磁共振方案，促进斑马鱼用于PKD研究。在该提案完成后，我们预计将显著加强父母的提案，旨在建立一种基于F0的遗传筛选方法，系统地识别ADPKD的新修饰基因。这一新型动物模型的推出有望大大推进ADPKD领域的发展。

ADPKD patients exhibit highly variable phenotypes, of which genetic modifiers were thought to play an important role. However, the identity of the genetic modifiers remains largely unknown, because of the lack of an efficient method. In this context, here, we propose to establish adult zebrafish as a new animal model that enables rapid and systematic discovery of genetic modifiers of ADPKD. We have generated substantial preliminary data for this proposal: 1) proving the molecular conservation of the zebrafish model by identifying corresponding homologues for 99% of the 82 PKD-associated genes; 2) developing zebrafish mutants for pkd1, pkd2 and ift140, 3 of the 6 known ADPKD-associated genes, which all lead to renal cyst formation; 3) demonstrating the feasibility of utilizing a novel microhomology-mediated end joining (MMEJ)-based F0 genetic method for screening ADPKD modifier genes. These preliminary data prompted us to raise a central hypothesis predicting that zebrafish is a conserved and efficient in vivo vertebrate model for discovering genetic modifiers of ADPKD. To test this hypothesis, we proposed the following three specific aims. In Aim 1, we will further prove the feasibility of our strategy by validating 2 novel protective ADPKD modifiers emerged from a MMEJ-based pilot F0 genetic screen. In Aim 2, we will explore the feasibility of using zebrafish models to assess ADPKD modifiers suggested from human patient population. In Aim 3, we will facilitate the use of zebrafish for PKD studies by further developing NMR protocols for the measurement of zebrafish kidney volume and cysts. Upon completion of the proposal, we anticipate to significantly strengthen the parent proposal, aiming to establish a F0-based genetic screen approach for systematically identifying new modifier genes for ADPKD. The launch of this novel animal model is expected to significantly advance the ADPKD field.