Metabolic consequences of cystinuria and genome engineering therapeutics

摘要 半胱氨酸尿症是一种遗传性人类肾脏疾病，发病率很高，每7000名退伍军人中就有一人受到影响。这个 这种疾病是由参与肾脏胱氨酸转运的基因突变导致尿胱氨酸升高引起的。 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，胱氨酸尿症患者会患上更多的慢性肾脏疾病和 高血压比其他结石的形成要好。此外，人们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)。这些额外的新陈代谢后果 胱氨酸尿症可能有助于观察到胱氨酸尿症患者发展为更多的慢性肾脏和 高血压与其他肾结石形成者相比。确定了疾病的遗传基础(突变 在SLC3a1，I型半胱氨酸尿症)中，存在靶向分子治疗的机会。在我们之前的基础上 Grant，我们提出了一种创新的实验设计来证明长期的表型纠正 使用转座子、腺相关病毒(AAV)和联合使用的完整动物的胱氨酸尿 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。 损伤与高血压的发展。我们将采用缺血再灌注损伤模型和单侧 输尿管梗阻评估对肾损伤的敏感性和恢复情况。我们还将评估α是否- 硫辛酸对这些代谢结果有任何影响，除了它已知的增加溶解度的能力之外 减少尿中的胱氨酸，防止胱氨酸结石的形成。在具体目标2中，我们建议设计一种嵌合体 我们将与CRISPR/Cas9进行比较，发现该转座酶能够挽救Slc3a1的表达 在缺乏SLC3a1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 包括转座子在内的多种基因组工程技术在体内永久性纠正胱氨酸尿症 伴随免疫抑制的技术、AAV-转座子杂交技术和CRISPR/CAS9 中介基因组编辑或定向整合。我们将尝试在新生和成年小鼠身上进行纠正 缺乏降低尿胱氨酸水平、升高血浆胱氨酸水平的SLC3a1检测，以及 胱氨酸结石减少术。拟议的研究将导致对新陈代谢的更好的理解 胱氨酸尿症的后果，并开发针对胱氨酸尿症和其他潜在疾病的基因组工程方法 影响退伍军人的肾脏疾病。

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.