Kidney specific site-directed integration for cystinuria

描述（由申请人提供）： 胱氨酸尿症是一种遗传性肾脏疾病，发病率高，每7000名退伍军人中就有1人患病。这种疾病是由参与肾胱氨酸转运的基因突变引起的，导致尿胱氨酸升高和肾结石形成。预防胱氨酸结石形成的医学治疗不是很有效，并且具有令人不快的副作用。随着疾病的遗传基础的确定（SLC 3A 1突变，胱氨酸尿症I型），靶向分子治疗的新机会存在。转座子是基于非病毒质粒DNA的载体，其代表用于染色体转基因插入和建立持续基因表达的有前景的新兴工具。为了使基于转座子的技术介导用于胱氨酸尿症的有效且安全的治疗性基因转移，肾靶向基因递送必须与能够将治疗性基因靶向整合至独特染色体元件的策略相结合，从而限制基因转移赋予的遗传毒性。在具体目标1中，我们开发了一种用于肾脏特异性DNA递送的新型流体动力学注射方法，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，并评估转座子整合位点的表达。 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。到目前为止，遗传性肾病的长期表型校正尚未证明使用基因转移。由SLC 3A 1编码的二元氨基酸转运蛋白的突变是胱氨酸尿症的最常见原因。在具体目标2中，我们提出使用转座子介导的肾脏特异性基因转移来表型地纠正SLC 3A 1-/-小鼠中的胱氨酸尿症。我们最近已经证明了通过将高度位点特异性的锌指蛋白（ZFP）融合到piggyBac转座酶上来操纵piggyBac转座子系统的染色体整合位点选择的能力，从而指导整合到用户选择的染色体元件中。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SLC 3A 1递送到培养的近端肾小管肾细胞中。此外，我们还建议建立一种动物模型，用于体内评价定点整合。这些研究的目的是证明转座子介导的基因转移的潜在治疗退伍军人肾综合征的能力。

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.