Kidney-targeted gene delivery for cystinosis

描述(申请人提供)：胱氨酸病是一种代谢遗传性疾病，其特征是细胞内胱氨酸积聚。受影响的人通常在一岁前出现近端小管病变(范科尼综合征)，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功能衰竭。胱氨酸积聚最终导致多器官功能障碍。药物半胱胺降低了细胞内的半胱氨酸浓度。然而，需要有规律的间隔剂量和一些不良的副作用使其难以管理。此外，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的基因治疗策略：逆行肾静脉注射表达功能性CTN的自补充腺相关病毒(ScAAV)，以治疗和预防膀胱病的近端小管病变和肾脏缺陷的进展。作为临床前研究，我们将使用CTNS-/-小鼠模型来治疗胱氨酸病。这些动物在所有被测试的器官中积累半胱氨酸和半胱氨酸晶体，并出现类似于在受影响的人类中观察到的肾脏损伤。AAV在肾脏中的基因转导研究很少，大多数AAV在体内和体外都使用AAV 2型来转导肾细胞。然而，AAV2的中和抗体在人群中的流行率非常高，可能会影响其基因传递的效率。因此，在具体目标1中，我们建议通过检测几种具有转导肾脏细胞的潜力和人类低中和抗体流行率的AAV血清型来优化通过肾静脉注射进行肾脏靶向基因传递的方法。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将用于这一目的，并将使用共聚焦显微镜、流式细胞术和定量PCR检测GFP以及IVIS成像系统检测荧光素酶的表达。在特定的目标2中，我们建议以肾静脉注射scAAV-CTN为基础来测试这种方法，作为一种治疗膀胱癌肾功能障碍的微创方法。肾功能将通过血液和尿液分析来测量，肾脏结构将通过组织学来测量。在一年的时间内，将在不同的时间点测量肾脏中的胱氨酸含量和CTNS的表达。这种定向基因疗法的免疫反应和安全性也将得到测试。这项工作代表了第一个针对胱氨酸病的基因治疗策略，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 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.