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

• 批准号: 10265368
• 负责人: MATTHEW H WILSON
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10265368
• 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

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 Cystinuria是一种遗传性的人类肾脏疾病，发病率明显影响7000名退伍军人。这 疾病是由参与肾胱氨酸转运的基因突变引起的，导致尿尿升高 与肾结石形成。从历史上看，囊这尿症的主要临床问题是胱氨酸溢出物 进入导致肾结石病的尿液。但是，囊肿性患者患上更多的慢性肾脏疾病和 高血压比其他石材形成器。此外，关于氨基丧失的其他后果知之甚少 酸转运功能（胱氨酸，鸟氨酸，赖氨酸和精氨酸的）。这些其他代谢后果 囊肿性可能有助于观察到囊肿患者患有更多慢性肾脏和 与其他肾结石的高血压相比。与定义的疾病的遗传基础（突变 在SLC3A1，I型Cystinuria）中，存在靶向分子疗法的机会。在我们以前的 Grant，我们提出了一种创新的实验设计，以证明长期表型校正 完整动物中使用转座子，腺相关病毒（AAV）和 CRISPR/CAS9基因组工程技术。 在特定目标1中，我们将评估代谢变化对肾脏敏感性和恢复的影响 伤害和高血压的发展。我们将使用缺血再灌注损伤模型和单侧 输尿管异议评估对肾脏损伤的敏感性和恢复。我们还将评估α-是否 lipoic酸对这些代谢后果有任何影响，而不是其已知的提高溶解度的能力 尿液中的胱氨酸并防止胱氨酸石的形成。在特定目标2中，我们建议设计嵌合 PiggyBac转座酶能够营救SLC3A1表达，我们将将其与CRISPR/CAS9进行比较 在缺乏SLC3A1的小鼠近端核细胞中介导的靶向整合。我们还建议尝试 通过多种基因组工程技术（包括转座子）在体内对囊肿的永久校正 与伴随的免疫抑制，混合AAV-Transposon技术和CRISPR/CAS9的技术 介导的基因组编辑或靶向整合。我们将尝试在新生儿和成年小鼠中进行纠正 缺乏SLC3A1分析可降低尿液中胱氨酸水平的分析，血浆中的胱氨酸水平增加和 胱氨酸结石的还原。拟议的研究将导致对代谢有更多的了解 囊这尿症的后果并开发了用于囊肿的基因组工程方法以及其他可能的其他 影响退伍军人的肾脏疾病。