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Proximal Tubule Targeted Gene Therapy for Cystinuria

胱氨酸尿症的近端小管靶向基因治疗

基本信息

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
项目类别：
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

项目摘要

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.

项目概要胱氨酸尿症等单基因肾病具有明确的遗传特征，但缺乏安全有效的方法临床治疗。胱氨酸尿症患者由于以下原因在尿路中形成大量基于胱氨酸的结石：肾近曲小管细胞无法重吸收胱氨酸，导致高达 70% 的病例发生慢性肾病。最常见的胱氨酸尿亚型 A 型是 SLC3A1 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。最近的进展基因组工程领域为单基因疾病提供了潜在的治疗方法包括胱氨酸尿症。当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克服这些疾病的理想模型疾病由于近端肾小管在肾脏内是可靶向的，因此低水平的 rBAT 估计可以预防结石形成和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，先前的工作已显示使用piggyBac转座子的转基因的有利整合效率系统。使用piggyBac转座子进行体内模型的肾脏靶向基因组工程可以这是通过一种新型近端小管靶向腺相关病毒（AAV）完成的。我假设肾特异性 AAV 与 SLC3A1 的 PiggyBac 转座子整合相结合将导致稳定的肾- 胱氨酸尿症模型的靶向表型校正。为了验证这个假设，我将设计肾脏特异性 AAV 载体在 AIM 1 中包含 PiggyBac-SLC3A1。自我互补的 AAV 已显示出改善的肾脏特异性，但其紧凑的尺寸需要将 SLC3A1 分成两个 AAV。因此，我将设计一个双AAV 使用同源重组和 mRNA 在体内重组表达全长 SLC3A1 的系统拼接。我还将在体外测试双 AAV-piggyBac-SLC3A1 系统的重组和功能。在目标 2，我将生成源自人类诱导多能干细胞 (iPSC) 的 SLC3A1-/- 肾脏类器官。然后，我将在交付所提议的系统后量化 rBAT 和胱氨酸运输的表达。最后，我会评估 AAV-piggyBac-SLC3A1 通过预防胱氨酸来纠正胱氨酸尿表型的潜力 AIM 3 中 A 型胱氨酸尿症小鼠模型中的结石形成。