Metabolic consequences of cystinuria and genome engineering therapeutics

摘要 胱氨酸尿症是一种遗传性人类肾脏疾病，发病率高，每7000名退伍军人中就有1人患病。的 疾病是由参与肾胱氨酸转运的基因突变引起的，导致尿胱氨酸升高 肾结石的形成。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，胱氨酸尿症患者发展为更多的慢性肾病， 高血压比其他结石形成者。此外，对氨基丢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）。这些额外的代谢后果 胱氨酸尿症可能有助于观察到胱氨酸尿症患者发生更多的慢性肾功能衰竭， 高血压与其他肾结石形成者相比。随着疾病的遗传基础的定义（突变 在Slc 3a 1，胱氨酸尿症I型）中，存在靶向分子治疗的机会。基于我们之前的 格兰特，我们提出了一个创新的实验设计，以证明长期的表型校正 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。 损伤和高血压的发展。采用缺血再灌注损伤模型， 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α- 除了已知的增加溶解度的能力之外， 尿中的胱氨酸，防止胱氨酸结石的形成。在具体目标2中，我们提出工程化嵌合体， piggyBac转座酶能够拯救Slc 3a 1表达，我们将其与CRISPR/Cas9进行比较 在缺乏Slc 3a 1的小鼠近端肾小管细胞中介导的靶向整合。我们还建议尝试 通过包括转座子在内的多种基因组工程技术在体内永久纠正胱氨酸尿症 伴随免疫抑制的技术、杂合AAV转座子技术和CRISPR/Cas9 介导的基因组编辑或靶向整合。我们将尝试在新生小鼠和成年小鼠中进行校正 缺乏Slc 3a 1测定尿中胱氨酸水平的降低、血浆中胱氨酸水平的增加，和 减少胱氨酸结石。拟议的研究将导致更好地了解代谢 胱氨酸尿症的后果，并开发胱氨酸尿症的基因组工程方法和潜在的其他 影响退伍军人的肾病

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.