Proximal Tubule Targeted Gene Therapy for Cystinuria

胱氨酸尿症的近端小管靶向基因治疗

• 批准号: 10534384
• 负责人: Jennifer Peek
• 金额: $ 3.19万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10534384
• 关键词: Amino Acid Transporter; Amino Acids; CRISPR/Cas technology; Cell Line; Cells; Chronic Kidney Failure; Clinical Treatment; Cystine; Cystinuria; Dependovirus; Disease model; Embryo; Engineering; Epithelial Cells; Evaluation; Failure; Fibroblasts; Genes; Genetic Recombination; Genome; Genome engineering; Goals; Health; Human; Hybrids; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Kidney; Kidney Diseases; Knock-out; Length; Life; Longevity; Mediating; Mendelian disorder; Messenger RNA; Modeling; Morbidity - disease rate; Mus; Mutation; Organoids; Paste substance; Pathogenicity; Patients; Phenotype; Prevention; Proximal Kidney Tubules; RNA Splicing; Specificity; Structure; System; Testing; Tissue Harvesting; Transcript; Transgenes; Urinary Calculi; Urinary tract; Work; adeno-associated viral vector; base; cell immortalization; cellular imaging; curative treatments; design; effective therapy; gene therapy; homologous recombination; improved; in vivo; in vivo Model; induced pluripotent stem cell; live cell imaging; mass spectrometric imaging; mortality; mouse genome; mouse model; novel; novel therapeutics; prevent; reconstitution; restoration; targeted delivery; transduction efficiency; transgene expression; urinary; vector

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.

项目摘要 单基因肾病如胱氨酸尿症在遗传学上有很好的特点，但缺乏安全有效的治疗方法 临床治疗。患有胱氨酸尿症的患者在他们的尿路中形成许多基于胱氨酸的结石， 肾近曲小管细胞不能重吸收胱氨酸，导致高达70%的慢性肾病。 胱氨酸尿症最常见的亚型A型是SLC 3A 1纯合缺陷的结果， 编码一种氨基酸转运蛋白（rBAT），可在近端小管中重吸收胱氨酸。的最新进展 基因组工程领域已经允许用于单基因疾病的潜在治愈性疗法 包括胱氨酸尿症。目前肾脏基因组工程的障碍包括递送和持续表达 转基因然而，胱氨酸尿症是研究和潜在地克服这些疾病的理想模型疾病。 由于近端小管是肾脏内的靶向屏障，估计低水平的rBAT可以防止结石 形成，胱氨酸尿症可以针对生命的任何阶段。当设计胱氨酸尿症的肾基因疗法时， 先前的工作已经显示了使用piggyBac转座子的转基因的有利整合效率 系统可以使用piggyBac转座子进行体内模型的肾靶向基因组工程改造。 用一种新的靶向近端小管的腺相关病毒（AAV）完成。我假设 肾特异性AAV与SLC 3A 1的piggyBac转座子整合的组合将导致稳定的肾- 胱氨酸尿症模型中的靶向表型校正。为了验证这一假设，我将设计肾脏特异性AAV 载体中含有piggyBac-SLC 3A 1。自身互补的AAV已经显示出改善的肾特异性， 但是其紧凑的尺寸需要将SLC 3A 1分成两个AAV。因此，我将设计一个双AAV 使用同源重组和mRNA在体内重组以表达全长SLC 3A 1的系统 拼接我还将在体外测试双重AAV-piggyBac-SLC 3A 1系统的重组和功能。在 在AIM 2中，我将产生来源于人诱导型多能干细胞（iPSC）的SLC 3A 1-/-肾类器官。 然后，我将定量表达rBAT和胱氨酸转运后交付的拟议系统。最后要 评估AAV-piggyBac-SLC 3A 1通过预防胱氨酸尿症表型纠正胱氨酸尿症的潜力 AIM 3中A型胱氨酸尿症小鼠模型中结石形成。