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：
10681248
负责人：
Jennifer Peek
金额：
$ 3.28万
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/10681248
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 absorption adeno-associated viral vector cell immortalization 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.

项目摘要诸如囊肿的单基因肾脏疾病在遗传上具有很好的特征，但缺乏安全有效临床治疗。囊肿性患者由于未能在肾脏近端细胞细胞中重新吸收胱氨酸，导致多达70％的病例导致慢性肾脏疾病。 A型A型的最常见亚类是SLC3A1的纯合缺陷的结果，该缺陷编码在近端管中重新吸收胱氨酸的氨基酸转运蛋白（RBAT）。最近的进步基因组工程领域允许对单基因疾病进行潜在的治疗治疗包括囊肿。当前的肾脏基因组工程障碍包括交付和持续表达翻译。然而，囊肿是一种理想的模型疾病，可以调查并可能克服这些疾病障碍物是近端小管是可靶向在肾脏内的，估计RBAT的水平低以防止石头形成和囊肿性可以在生命的任何阶段。在设计肾上腺素肾疗法时，先前的工作显示了使用Piggybac Transposon的翻译的优势集成效率系统。使用Piggybac转座子进行体内模型的肾脏针对肾脏靶向的基因组工程可以是以一种新型的近端管靶腺相关病毒（AAV）完成。我假设肾脏特异性AAV与SLC3A1的Piggybac转座子整合的组合将导致稳定的肾脏 - 囊性模型中的靶向表型校正。为了检验这一假设，我将设计肾脏特异性AAV 在AIM 1中包含PiggyBac-Slc3a1的向量。自我平均AAV已显示出改善的肾脏特异性，但是，将SLC3A1分成两个AAV所需的紧凑尺寸。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SLC3A1的系统剪接。我还将在体外测试双重AAV-piggybac-SLC3A1系统的重组和功能。在 AIM 2，我将生成源自人诱导多能干细胞（IPSC）的SLC3A1 - / - 肾脏器官。然后，我将在交付拟议的系统后量化RBAT和Cystine转运的表达。最后，我会的通过预防Cystine评估AAV-piggybac-SLC3A1在表型上纠正囊肿的潜力在AIM 3中的A型囊肿型小鼠模型中的石材形成。