Toxicology studies for gene-modified 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。受影响的个人通常表现为 近端肾小管病变在一岁之前，最终发展为终末期肾功能衰竭。胱氨酸蓄积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含量。然而，半胱胺不能预防近端肾小管病变和终末期肾功能衰竭，只会延缓疾病的进展。该项目的长期目标是开发一种新的治疗膀胱癌的方法，即通过移植体外修饰的自体造血干细胞和祖细胞(HSPC)来表达具有功能的CTNS基因。利用CTNS-/-小鼠模型，我们发现，表达CTNS的同基因Sca1+HSPC移植导致大量的骨髓来源细胞植入，并显著降低了所有受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进展。我们使用我们的慢病毒载体构建的PCCL-CTNS在体外转导HSPC获得了相同的结果，并在CTNS-/-小鼠中建立了第一个概念验证，即这种策略可以在疾病显著进展之前对患有胱氨酸病的年轻患者起作用。在获得FDA批准进入Pre-Investigator新药(IND)申请后，我们现在提议进行药理学/毒理学研究，以获得针对胱氨酸病的I期临床试验的IND。这种治疗将是一种终生的治疗方法，可以预防肾移植和与胱氨酸病相关的长期并发症。慢病毒载体已经证明了它在小鼠和人类身上长期转导HSPC的有效性。所有的药理学/毒理学研究都将在临床前使用根据《良好制造规范》生产的载体PCCL-CTN进行。在具体目标1中，我们建议使用我们的载体PCCL-CTN优化CD34+细胞的转导，并使用克隆形成单位(CFU)试验测试转导的细胞通过谱系承诺的祖细胞而不会变成白血病的能力。另一种评估整合病毒载体遗传毒性的体外试验是体外永生化(IVIM)试验，该试验使用 小鼠血统阴性骨髓细胞。载体拷贝数(VCN)和载体整合位点(VI)将在两种检测的最终克隆和克隆中确定。在特定的目的2中，我们将用PCCL-CTNS体外转导的小鼠Sca1+HSPC在体内测试这一策略的有效性和安全性，并将其移植到原发和继发受体CTNS-/-小鼠体内。疗效将通过CTNS在血液和组织样本中的表达、组织胱氨酸水平和肾功能来衡量。毒性将通过全面的临床和组织学分析，通过评估髓系和淋巴样细胞中的VCN和VIS，以及检测潜在的CTNS蛋白抗体免疫来确定。这项工作代表了胱氨酸病I期临床试验的最后一步，也是治疗其他溶酶体储存障碍的概念验证。

DESCRIPTION (provided by applicant): Cystinosis is a metabolic hereditary disease characterized by intracellular accumulation of cystine. Affected individuals typically present with proximal tubulopathy before one year of age and eventually progress to end- stage renal failure. Cystine accumulation leads to multi-organ dysfunction. The drug cysteamine reduces the intracellular cystine content. However, cysteamine does not prevent the proximal tubulopathy nor the end- stage renal failure and only delay the progression of the disease. The long-term objective of this project is to develop a new treatment for cystinosis by transplantation of autologous Hematopoietic Stem and Progenitor Cells (HSPC) genetically modified ex vivo to express a functional CTNS gene. Using the mouse model for cystinosis, the Ctns-/- mice, we showed that transplantation of syngeneic Sca1+ HSPC expressing Ctns resulted in abundant bone marrow-derived cell engraftment and significant reductions of cystine content in all the tissues tested. This treatment also prevented the progression of kidney dysfunction. We obtained the same results with ex vivo transduced HSPC using our lentiviral vector construct, pCCL-CTNS, and established the first proof-of-concept in the Ctns-/- mice that this strategy could work in young patients with cystinosis before significant disease progression. After obtaining the approval from the FDA to move forward to a pre- Investigator New Drug (IND) application, we are now proposing the pharmacology/toxicology studies required to obtain an IND for a phase I a clinical trial for cystinosis. This treatment would represent a life-long theray that may prevent kidney transplantation and long-term complications associated with cystinosis. Lentiviral vectors have proven its efficacy for long-term HSPC transduction in mice but also in humans. All the pharmacology/toxicology studies will be done with a pre-clinical batch of the vector pCCL-CTNS produced under Good Manufacturing Practice. In Specific aim 1, we propose to optimize the transduction of human CD34+ cells using our vector pCCL-CTNS and to test the capacity of the transduced cells to go through lineage-committed progenitors without becoming leukemic using the Colony-Forming Units (CFU) assay. The other in vitro assay to assess genotoxicity of integrating viral vectors is the In Vitro Immortalization (IVIM) assay using murine lineage-negative bone marrow cells. Vector Copy Numbers (VCN) and Vector Integration Sites (VIS) will be determined in the final colonies and clones for both assays. In Specific aim 2, we will test the efficacy and safety of this strategy in vivo using murine Sca1+ HSPC ex vivo transduced by pCCL-CTNS and transplanted in primary and secondary recipient Ctns-/- mice. Efficacy will be measured by CTNS expression in blood and tissue samples, tissue cystine levels and renal function. Toxicity will be determined by comprehensive clinical and histological tissue analyses, by assessing VCN and VIS in myeloid and lymphoid cells and detecting potential antibody immunity to CTNS proteins. This work represents the last steps towards a phase I clinical trial for cystinosis and is also a proof of concept to treat other lysosomal storage disorders.