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）： 摘要囊这尿症是一种遗传性的人肾脏疾病，有明显的发病率影响了7000名退伍军人中有1个。该疾病是由参与肾胱氨酸转运的基因突变引起的，导致尿胱氨酸和肾结石的形成升高。防止形成胱氨酸石的药物治疗不是很有效，并且具有不愉快的副作用。由于疾病定义的遗传基础（SLC3A1中的突变，I型的突变），存在靶向分子疗法的新机会。转座子是非病毒质粒-DNA的非病毒质粒载体，代表有希望的新兴工具用于染色体转基因插入和持续基因表达的建立。为了使基于转座子的技术介导有效且安全的治疗基因转移囊肿性，必须将靶向肾脏的基因递送与能够靶向将治疗基因整合到独特的染色体元素中的策略相结合，从而限制了基因转移赋予的基因毒性。在特定目标1中，我们开发了一种新型的流体动力注射方法，用于肾脏特异性DNA递送，并证明了转座子能够使用该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的长期基因表达程度，进一步阐明稳定转染的肾细胞类型，分析体内基因转移后转座子整合位点，并评估 转座子介导的基因转移到完整肾脏后短期和长期毒性的可能性。迄今为止，尚未使用基因转移证明对遗传性肾脏疾病的长期表型矫正尚未证明。由SLC3A1编码的二骨氨基酸转运蛋白的突变是囊这尿症的最常见原因。在特定的目标2中，我们建议在SLC3A1 - / - 小鼠中使用转座子介导的肾脏特异性基因转移到表型上正确的囊肿。最近，我们证明了通过将高度位点特异性的锌指蛋白（ZFP）融合到PiggyBac转座酶，从而将整合引导到用户选择的染色体元素中，从而操纵Piggybac Transposon系统的染色体整合位点选择。在特定的目标3中，我们提出了一种创新的方法来选择现场定向的事件，并将以位于位置的方式将SLC3A1传递到培养的近端管状肾细胞中。在Additon中，我们建议开发一种用于评估体内位置定向整合的动物模型。这些研究旨在证明转座子介导的基因转移对退伍军人的潜在肾脏综合征的能力。