Kidney-targeted gene delivery for cystinosis

描述（由申请人提供）：胱氨酸病是一种代谢性遗传性疾病，其特征为胱氨酸在细胞内蓄积。 受影响的个体通常在一岁之前出现近端小管病变（范可尼综合征），并且在第一个十年结束时没有特定的治疗进展到终末期肾衰竭。胱氨酸蓄积最终导致多器官功能障碍。药物半胱胺降低胱氨酸的细胞内浓度。然而，需要规律间隔的剂量和许多不期望的副作用使得其施用困难。半胱胺不能预防近端肾小管病变或终末期肾功能衰竭.该项目的长期目标是开发一种肾脏特异性基因治疗策略：逆行肾静脉注射表达功能性CTNS的自身互补腺相关病毒（scAAV），以治疗和预防近端小管病变和胱氨酸病中肾缺陷的进展。作为临床前研究，我们将使用Ctns-/-鼠模型进行胱氨酸病研究。这些动物在所有检测的器官中积累胱氨酸和胱氨酸晶体，并发生与受影响人类中观察到的肾损伤相似的肾损伤。关于AAV在肾脏中进行基因递送的研究很少，大多数研究使用AAV血清型2在体内和体外转染肾细胞。然而，人群中AAV 2的中和抗体的流行率非常高，可能会影响其基因递送的效率。因此，在具体目标1中，我们提出通过测试几种AAV血清型来优化经由肾静脉注射的肾靶向基因递送，所述AAV血清型具有转导肾细胞的潜力并且在人中中和抗体的流行率低。还将确定最佳剂量。表达绿色荧光蛋白（GFP）和荧光素酶报告基因的scAAV将用于该目的，并且表达将使用共聚焦显微镜、流式细胞术和定量PCR（GFP）以及IVIS成像系统（荧光素酶）进行可视化和定量。在具体目标2中，我们提出基于肾静脉注射scAAV-CTNS来测试这种方法，作为用于治疗胱氨酸病中的肾功能障碍的微创手术。将通过血液和尿液分析测量肾功能，通过组织学测量肾结构。将在一年期间的不同时间点测量肾脏中的胱氨酸含量和CTNS表达。这种定向基因治疗的免疫应答和安全性也将被测试。这项工作代表了胱氨酸病的第一个基因治疗策略，并为未来的临床试验奠定了基础。它也代表了对其他遗传性肾病的肾脏特异性治疗的概念证明。

DESCRIPTION (provided by applicant): Cystinosis is a metabolic hereditary disease characterized by intracellular accumulation of cystine. Affected individuals typically present with proximal tubulopathy (Fanconi syndrome) before one year of age and without specific treatment progress to end-stage renal failure by the end of the first decade. Cystine accumulation eventually leads to multi-organ dysfunction. The drug cysteamine reduces the intracellular concentration of cystine. However, the need for regularly spaced doses and a number of undesirable side effects render its administration difficult. Moreover, cysteamine does not prevent the proximal renal tubulopathy or the end- stage renal failure. The long-term objective of this project is to develop a kidney-specific gene therapy strategy: retrograde renal vein injection of self-complementary adeno-associated virus (scAAV) expressing a functional CTNS to treat and prevent the proximal tubulopathy and progression of renal defects in cystinosis. As pre-clinical studies, we will use the Ctns-/- murine model for cystinosis. These animals accumulate cystine and cystine crystals in all organs tested and develop kidney injuries similar to those observed in affected humans. Few studies have been performed on AAV for gene delivery in the kidney and most of them used AAV serotype 2 to transduce renal cells in vivo and in vitro. However, the prevalence of neutralizing antibodies in the human population for AAV2 is very high and would probably impact its efficiency for gene delivery. Therefore, in Specific aim 1, we propose to optimize kidney-targeted gene delivery via renal vein injection by testing several AAV serotypes that have the potential of transducing renal cells and a low prevalence of neutralizing antibodies in human. The optimal dose will be also determined. scAAV expressing the green fluorescent protein (GFP) and luciferase reporter genes will be used in this aim and expression will be visualized and quantified using confocal microscopy, flow cytometry and quantitative PCR for GFP, and IVIS imaging system for luciferase. In Specific aim 2, we propose to test this approach based on renal vein injection of scAAV-CTNS as a minimally invasive procedure for treating the renal dysfunction in cystinosis. Renal function will be measured by blood and urine analyses and renal structure by histology. Cystine content and CTNS expression will be measured in the kidney at different time points during a one-year period. The immune response and safety of this directed gene therapy will also be tested. This work represents the first gene therapy treatment strategies for cystinosis and builds the foundations for a future clinical trial. It also represents a proof of concept for a kidney-specific therapy for other hereditary nephropathies.