Metabolic consequences of cystinuria and genome engineering therapeutics

胱氨酸尿症和基因组工程疗法的代谢后果

基本信息

批准号：
9898319
负责人：
MATTHEW H WILSON
金额：
--
依托单位：
VETERANS HEALTH ADMINISTRATION
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9898319
关键词：
Adult Affect Amino Acids Animals Arginine Biological Assay Blood Blood Pressure CRISPR/Cas technology Cells Chronic Chronic Kidney Failure Clinical Complementary DNA Cystine Cystinuria DNA Dependovirus Development Diet Disease Engineering Exhibits Experimental Designs Funding Gene Delivery Gene Expression Gene Mutation Gene Targeting Gene Transfer Genes Genetic Genome Genome engineering Grant Guide RNA Health Human Hybrids Hypertension Immunosuppression In Vitro Inherited Injury to Kidney Kidney Kidney Calculi Kidney Diseases Knock-out Liver Lysine Mediating Medical Metabolic Methods Mission Modeling Morbidity - disease rate Mus Mutation Neonatal Nephrolithiasis Ornithine Patients Phenotype Plague Plasma Proteins Publishing Recovery Reduced Glutathione Reperfusion Injury Research Solubility System Technology Technology Transfer Testing Therapeutic Thioctic Acid Transcription Coactivator Transposase Tubular formation Ureteral obstruction Urine Variant Veterans adeno-associated viral vector amino acid metabolism base gene correction genome editing human disease improved in vivo innovation molecular targeted therapies mouse model non-viral gene delivery prevent response side effect urinary vector

项目摘要

ABSTRACT Cystinuria is an inherited human renal disease with significant morbidity affecting 1 in 7000 veterans. The disease is caused by mutation of genes involved in renal cystine transport resulting in elevated urinary cystine with kidney stone formation. Historically, the primary clinical concern of cystinuria has been that cystine spills into the urine resulting in nephrolithiasis. However, cystinuria patients develop more chronic kidney disease and hypertension than other stone formers. Additionally, little is known about other consequences of loss of amino acid transport function (of cystine, ornithine, lysine, and arginine). These additional metabolic consequences of cystinuria likely contribute to the observations that cystinuria patients develop more chronic kidney and hypertension compared to other kidney stone formers. With the genetic basis of the disorder defined (mutation in Slc3a1, cystinuria type I), opportunities for targeted molecular therapies exist. Building upon our previous grant, we propose an innovative experimental design to demonstrate long-term phenotypic correction of cystinuria in an intact animal using a combination of transposon, adeno-associated virus (AAV), and CRISPR/Cas9 genome engineering technologies. In specific aim 1, we will evaluate the effects of the metabolic changes on sensitivity to and recovery from kidney injury and the development of hypertension. We will use ischemia reperfusion injury models and the unilateral ureteral obstruction to evaluate sensitivity to and recovery from kidney injury. We will also evaluate whether α- lipoic acid has any effect on these metabolic consequences other than its known ability to increase the solubility of cystine in the urine and prevent cystine stone formation. In specific aim 2, we propose to engineer a chimeric piggyBac transposase capable of rescuing of Slc3a1 expression and we will compare this to CRISPR/Cas9 mediated targeted integration in mouse proximal tubular cells lacking Slc3a1. We also propose to attempt permanent correction of cystinuria in vivo by multiple genome engineering technologies including transposon technology with concomitant immunosuppression, hybrid AAV-transposon technology and CRISPR/Cas9 mediated genome editing or targeted integration. We will attempt correction both in neonatal and adult mice lacking Slc3a1 assaying for reduction of cystine level in the urine, increase of cystine level in the plasma, and reduction of cystine stones. The proposed studies will lead to a greater understanding of the metabolic consequences of cystinuria and develop genome engineering approaches for cystinuria and potentially other kidney diseases affecting veterans.

抽象的胱氨酸尿症是一种遗传性人类肾脏疾病，每 7000 名退伍军人中就有 1 人发病率很高。该疾病是由参与肾脏胱氨酸转运的基因突变引起的，导致尿胱氨酸升高从历史上看，胱氨酸尿症的主要临床问题是胱氨酸溢出。进入尿液导致肾结石，然而，胱氨酸尿症患者会患上更多的慢性肾脏疾病。与其他结石形成者相比，高血压更严重，此外，人们对氨基酸损失的其他后果知之甚少。酸转运功能（胱氨酸、鸟氨酸、赖氨酸和精氨酸）。胱氨酸尿症可能有助于观察到胱氨酸尿症患者会出现更多的慢性肾病和肾病。与其他肾结石形成者相比，高血压具有确定的疾病遗传基础（突变）。在 Slc3a1（I 型胱氨酸尿症）中，基于我们之前的研究，存在靶向分子治疗的机会。授予，我们提出了一种创新的实验设计来证明长期表型校正使用转座子、腺相关病毒 (AAV) 和 CRISPR/Cas9基因组工程技术。在具体目标 1 中，我们将评估代谢变化对肾脏敏感性和恢复的影响我们将使用缺血再灌注损伤模型和单侧。输尿管梗阻以评估肾损伤的敏感性和恢复情况，我们还将评估是否α-。除了已知的增加溶解度的能力之外，硫辛酸对这些代谢结果还有任何影响在具体目标 2 中，我们建议设计一种嵌合体。 PiggyBac 转座酶能够挽救 Slc3a1 表达，我们将其与 CRISPR/Cas9 进行比较我们还建议尝试在缺乏 Slc3a1 的小鼠近端肾小管细胞中介导靶向整合。通过包括转座子在内的多种基因组工程技术永久纠正体内胱氨酸尿症伴随免疫抑制技术、混合 AAV 转座子技术和 CRISPR/Cas9 我们将尝试在新生和成年小鼠中进行介导的基因组编辑或靶向整合。缺乏尿液中胱氨酸水平降低、血浆中胱氨酸水平升高的 Slc3a1 检测，以及减少胱氨酸结石的研究将加深对代谢的了解。胱氨酸尿症的后果并开发针对胱氨酸尿症和潜在其他疾病的基因组工程方法影响退伍军人的肾脏疾病。