Proximal Tubule Targeted Gene Therapy for Cystinuria

项目总结 单基因肾脏疾病，如胱氨酸尿症，在遗传学上有很好的特点，但缺乏安全性和有效性。 临床治疗。胱氨酸尿症患者在尿路中形成大量以胱氨酸为基础的结石，原因是 未能在肾近端小管细胞中重新吸收胱氨酸，导致高达70%的病例出现慢性肾脏疾病。 最常见的A型半胱氨酸尿症是SLC3A1纯合子缺陷的结果，它 编码一种氨基酸转运蛋白(RBAT)，可在近端小管重新吸收半胱氨酸。近几年来的研究进展 基因组工程领域已经允许对单基因疾病进行潜在的根治疗法 包括半胱氨酸尿症。目前肾脏基因组工程的障碍包括传递和持续表达 转基因。然而，胱氨酸尿症是一种理想的研究和克服这些疾病的模型疾病。 屏障由于近端小管是肾脏内的靶点，估计低水平的rBAT可以防止结石 形成，半胱氨酸尿症可以针对生命的任何阶段。在设计针对胱氨酸尿症的肾脏基因疗法时， 以前的工作已经表明，使用iggyBac转座子具有良好的转基因整合效率 系统。在活体模型中使用iggyBac转座子进行肾脏靶向基因组工程 通过一种新型的近端小管靶向腺相关病毒(AAV)完成。我猜想 将肾脏特异性AAV与SLC3A1转座子整合的iggyBac转座子相结合，将导致稳定的肾脏- 胱氨酸尿症模型中的靶向表型纠正。为了验证这一假设，我将设计肾脏特异的AAV AIM 1.自身补体AAV表现出更高的肾脏特异性 但其紧凑的尺寸需要将SLC3A1分成两个AAV。因此，我将设计一种双AAV 利用同源重组和mRNA在体内重组以表达全长SLC3A1的系统 拼接。我还将在体外测试双AAV-iggyBac-SLC3A1系统的重组和功能。在……里面 目的2，从人可诱导多能干细胞(IPSCs)中产生SLC3A1-/-肾器官。 然后，我将量化rBAT的表达和半胱氨酸运输后交付的建议系统。最后，我会 评估AAV-iggyBac-SLC3A1通过预防胱氨酸表型纠正胱氨酸尿症的潜力 A型半胱氨酸尿症小鼠模型的结石形成。

PROJECT SUMMARY Monogenic kidney diseases such as cystinuria are well characterized genetically, but lack safe and effective clinical treatments. Patients with cystinuria form numerous cystine-based stones in their urinary tract due to failure to reabsorb cystine in renal proximal tubule cells, leading to chronic kidney disease in up to 70% of cases. The most common subclass of cystinuria, type A, is a result of a homozygous deficiency of SLC3A1, which encodes an amino acid transporter (rBAT) that reabsorbs cystine in proximal tubules. Recent advances in the genome engineering field have allowed for potentially curative therapy for monogenic diseases including cystinuria. Current barriers to renal genome engineering include delivery and sustained expression of transgenes. However, cystinuria is an ideal model disease to investigate and potentially overcome these barriers as the proximal tubule is targetable within the kidney, a low level of rBAT is estimated to prevent stone formation, and cystinuria could be targeted at any stage of life. When designing renal gene therapy for cystinuria, previous work has shown advantageous integration efficiency of transgenes using the piggyBac transposon system. Kidney-targeted genome engineering using piggyBac transposons for in vivo models can be accomplished with a novel proximal tubule-targeted adeno-associated virus (AAV). I hypothesize that the combination of a renal specific AAV with piggyBac transposon integration of SLC3A1 will lead to stable, kidney- targeted phenotypic correction in models of cystinuria. To test this hypothesis, I will engineer renal-specific AAV vectors to contain piggyBac-SLC3A1 in AIM 1. Self-complementary AAV has shown improved kidney specificity, but its’ compact size necessitates the splitting of SLC3A1 into two AAVs. Therefore, I will design a dual AAV system that recombines in vivo to express full length SLC3A1 using homologous recombination and mRNA splicing. I will also test the recombination and functionality of the dual AAV-piggyBac-SLC3A1 system in vitro. In AIM 2, I will generate SLC3A1-/- kidney organoids derived from human inducible pluripotent stem cells (iPSCs). I will then quantify expression of rBAT and cystine transport after delivery of the proposed system. Finally, I will assess the potential of AAV-piggyBac-SLC3A1 to phenotypically correct cystinuria through prevention of cystine stone formation in a mouse model of type A cystinuria in AIM 3.