Lentiviral-transduced hematopoictic stem cell transplantation 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，以胱氨酸在细胞内积聚为特征。受影响的个体通常在一岁前出现近端肾小管病变（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。胱氨酸积累最终导致多器官功能障碍。药物半胱胺降低细胞内胱氨酸的浓度。然而，需要定期间隔的剂量和一些不良副作用使其施用困难。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体外基因修饰的自体造血干细胞移植来表达功能性CTNS基因，开发一种新的胱氨酸病治疗方法。作为临床前研究，我们将使用Ctns-/-小鼠模型来研究胱氨酸病。这些动物在所测试的所有器官中积累胱氨酸和胱氨酸晶体，并出现与在受影响的人类中观察到的类似的眼部变化、神经缺陷和肾脏损伤。我们的初步数据表明，移植表达Ctns的同基因全骨髓细胞（BMC）或纯化造血干细胞（HSC）可导致BMC或HSC衍生细胞的组织植入，并显著降低所有测试组织中的胱氨酸含量。这种治疗还可以防止肾功能障碍的发生和发展。我们现在建议使用从Ctns-/-小鼠中分离的Sca1+ HSC和慢病毒载体（LV）在体外传递Ctns基因。LV已经在小鼠和人类身上证明了其长期造血干细胞转导的有效性。在具体目标1中，我们提出利用表达LV的报告基因优化小鼠Sca1+ HSC的转导，以提高临床试验的安全性和效率，并显著降低成本。在特异性目的2中，我们将测试LV转导的、表达ctns的HSC是否可以预防年轻小鼠胱氨酸病介导的组织损伤，并可能逆转老年小鼠胱氨酸病介导的损伤。这些策略的有效性将通过测量不同组织区室中CTNS表达和胱氨酸含量以及通过完善的功能研究来测试预防或治疗肾功能障碍、眼异常、骨异常和神经缺陷来测试。该策略的免疫反应和安全性也将在Ctns-/-小鼠中进行测试，以及药物介导的骨髓消融的毒性。这项工作代表了胱氨酸病的第一个干细胞和基因治疗策略，并为未来的临床试验奠定了基础。这也代表了自体HSC移植治疗其他溶酶体贮积性疾病的概念证明。

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.