Kidney specific site-directed integration for cystinuria

肾脏特异性定点整合治疗胱氨酸尿症

• 批准号: 8542365
• 负责人: MATTHEW H WILSON
• 金额: --
• 依托单位: MICHAEL E DEBAKEY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-10-01 至 2017-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8542365
• 关键词: Adverse effects; Affect; Amino Acid Transporter; Animal Model; Animals; Area; Basic Amino Acids; Calculi; Cells; Cystine; Cystinuria; DNA Transposons; DNA delivery; Data; Diamino Amino Acids; Disease; Elements; Engineering; Epithelial Cells; Event; Experimental Designs; Future; Gene Delivery; Gene Expression; Gene Mutation; Gene Transfer; Genes; Genetic; Genomics; Goals; Health; Human; Image; Immunohistochemistry; Inherited; Injection of therapeutic agent; Kidney; Kidney Calculi; Kidney Diseases; Mediating; Medical; Methodology; Methods; Mission; Modeling; Molecular Target; Monitor; Morbidity - disease rate; Mus; Mutation; Nephrons; Plague; Proteins; Research; Research Design; Research Proposals; Site; Somatic Cell; Syndrome; System; Technology; Time; Tissues; Toxic effect; Transgenes; Transposase; Tubular formation; Veterans; Viral; Viral Vector; Work; Zinc Fingers; base; cell type; genotoxicity; improved; in vivo; innovation; kidney cell; novel; plasmid DNA; prevent; public health relevance; therapeutic gene; tool; urinary; vector

DESCRIPTION (provided by applicant): 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 and kidney stone formation. Medical treatments to prevent the formation of cystine stones are not very effective and have unpleasant side effects. With the genetic basis of the disorder defined (mutation in SLC3A1, cystinuria type I), new opportunities for targeted molecular therapies exist. Transposons are non- viral plasmid-DNA based vectors that represent promising emerging tools for chromosomal transgene insertion and establishment of persistent gene expression. In order for transposon-based technologies to mediate effective and safe therapeutic gene transfer for cystinuria, kidney-targeted gene delivery must be combined with strategies able to target integration of therapeutic genes to unique chromosomal elements thereby limiting genotoxicity imparted by gene transfer. In specific aim 1, we have developed a novel hydrodynamic injection method for kidney-specific DNA delivery and have demonstrated that transposons are capable of achieving long-term gene expression using this technology. We propose to evaluate for the extent of long-term gene expression after kidney specific gene transfer, further elucidate the kidney cell types stably transfected, analyze transposon integration sites after gene transfer in vivo, and evaluate for the possibility of short- and long-term toxicity after transposon- mediated gene transfer to intact kidney. Thus far, long-term phenotypic correction of an inherited kidney disease has not been demonstrated using gene transfer. Mutation of a dibasic amino acid transporter encoded by SLC3A1 is the most frequent cause of cystinuria. In specific aim 2, we propose to use transposon-mediated kidney-specific gene transfer to phenotypically correct cystinuria in SLC3A1 -/- mice. We have recently demonstrated the ability to manipulate chromosomal integration site selection of the piggyBac transposon system by fusing a highly site-specific zinc finger protein (ZFP) to the piggyBac transposase, thereby directing integration into user-selected chromosomal elements. In specific aim 3, we propose an innovative method of selecting for site-directed events and will deliver SLC3A1 in a site-directed manner into cultured proximal tubular kidney cells. In additon, we propose to develop an animal model for evaluating for site-directed integration in vivo. These studies are intended to demonstrate the capability of transposon-mediated gene transfer for potential treatment renal syndromes in veterans.

描述(由申请人提供)： 摘要胱氨酸尿症是一种遗传性人类肾脏疾病，发病率极高，每7000名退伍军人中就有一人患病。这种疾病是由参与肾胱氨酸运输的基因突变引起的，导致尿胱氨酸升高和肾结石形成。防止胱氨酸结石形成的药物治疗效果不是很好，而且有令人不快的副作用。随着疾病的遗传基础被确定(SLC3A1突变，I型胱氨酸尿症)，靶向分子治疗的新机会出现了。转座子是一种基于非病毒载体的载体，代表了染色体转基因插入和建立持久基因表达的新兴工具。为了使基于转座子的技术能够有效和安全地介导针对胱氨酸尿症的治疗性基因转移，肾脏靶向基因传递必须与能够靶向将治疗基因整合到独特的染色体元件的策略相结合，从而限制基因转移所带来的遗传毒性。在具体目标1中，我们开发了一种用于肾脏特异性DNA传递的新型流体动力注射方法，并证明了转座子能够利用这一技术实现长期基因表达。我们建议评估肾脏特异性基因转移后长期基因表达的程度，进一步阐明稳定转染的肾脏细胞类型，分析体内基因转移后转座子整合的位置，并评估 转座子介导的基因转移到完整肾脏后的短期和长期毒性的可能性。到目前为止，遗传性肾脏疾病的长期表型纠正还没有通过基因转移得到证实。由SLC3A1编码的二碱性氨基酸转运体突变是导致胱氨酸尿症的最常见原因。在特定目标2中，我们建议使用转座子介导的肾脏特异性基因转移来纠正SLC3A1-/-小鼠的表型半胱氨酸尿症。我们最近展示了通过将高度位点特异性的锌指蛋白(ZFP)融合到iggyBac转座酶上，从而指导整合到用户选择的染色体元件中，从而操纵iggyBac转座子系统的染色体整合位点选择的能力。在具体目标3中，我们提出了一种选择定点事件的创新方法，并将以定点方式将SLC3A1转移到培养的近端肾小管上皮细胞。此外，我们还建议建立一种动物模型来评估体内的定点整合。这些研究旨在证明转座子介导的基因转移对退伍军人肾脏综合征的潜在治疗能力。