Lentiviral-transduced hematopoictic stem cell transplantation 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通过移植体外转基因的自体造血干细胞来表达功能性CTNS基因，从而开发一种治疗胱氨酸病的新方法。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，并出现与在受影响的人类中观察到的相似的眼睛变化、神经缺陷和肾脏损伤。我们的初步数据显示，表达CTN的同基因全骨髓细胞(BMC)或纯化的造血干细胞(HSC)移植后，BMC或HSC来源的细胞在所有受试组织中的组织植入和胱氨酸含量显著降低。这种治疗还可以防止肾功能障碍的发展和进展。我们现在建议使用从CTNS-/-小鼠分离的Sca1+HSC和慢病毒载体(LV)在体外传递CTNS基因。LV已经证明了它对小鼠和人类的长期HSC转导的有效性。在具体目标1中，我们建议使用LV表达报告基因来优化小鼠Sca1+HSC的转导，以提高临床试验的安全性和效率，同时显著降低成本。在特定的目标2中，我们将测试LV转导的、表达CTNS的HSC是否可以防止幼年鼠的胱氨酸病介导的组织损伤，并有可能逆转老年鼠的胱氨酸病介导的损伤。这些策略的有效性将通过测量CTNS在不同组织中的表达和胱氨酸含量来测试，并通过成熟的功能研究来测试预防或治疗肾功能障碍、眼睛异常、骨骼异常和神经缺陷。这一策略的免疫反应和安全性也将在CTNS-/-小鼠身上测试药物介导的骨髓清除术的毒性。这项工作代表了首个针对胱氨酸病的干细胞和基因疗法治疗策略，并为未来的临床试验奠定了基础。这也代表了自体造血干细胞移植策略用于治疗其他溶酶体储存障碍的概念证明。

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 new treatment for cystinosis by transplantation of autologous hematopoietic stem cells genetically modified ex vivo to express a functional CTNS gene. 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 ocular changes, neurological defects and kidney injuries similar to those observed in affected humans. Our preliminary data showed that transplantation of syngeneic whole bone marrow cells (BMC) or purified hematopoietic stem cells (HSC) expressing Ctns resulted in tissue engraftment of BMC or HSC-derived cells and significant reductions of cystine content in all the tissues tested. This treatment also prevented the development and progression of kidney dysfunction. We now propose to use Sca1+ HSC isolated from Ctns-/- mice and lentiviral vector (LV) for delivering the CTNS gene ex vivo. LV has proven its efficacy for long-term HSC transduction in mice but also in humans. In Specific aim 1, we propose to optimize the transduction of murine Sca1+ HSC using LV expressing reporter genes to enhance the safety and efficiency of a clinical trial as well as significantly reduce costs. In Specific aim 2, we will test if LV- transduced, CTNS-expressing HSC can prevent cystinosis-mediated tissue injury in young mice and potentially reverse cystinosis-mediated injury in older mice. The efficiency of these strategies will be tested by measuring CTNS expression and cystine content in different tissue compartments and by well-established functional studies to test the prevention or treatment of the kidney dysfunction, eye anomalies, bone anomalies and neurological defects. The immune response and safety of this strategy will be also tested as well as the toxicity of drug-mediated myeloablation in Ctns-/- mice. This work represents the first stem cell and gene therapy treatment strategies for cystinosis and builds the foundations for a future clinical trial. It also represents a proof of concept for autotologous HSC transplantation strategy to treat other lysosomal storage disorders.