Toxicology studies for gene-modified stem cell transplantation for cystinosis

基因修饰干细胞移植治疗胱氨酸病的毒理学研究

• 批准号: 8715802
• 负责人: Stephanie Cherqui
• 金额: $ 26.97万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-06 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8715802
• 关键词: 12 year old; Adolescent; Adult; Adverse effects; Affect; Age-Years; Allogenic; Animals; Antibodies; Autologous Transplantation; Biological Assay; Blindness; Blood Cells; Blood specimen; Bone Marrow; Bone Marrow Cells; Bone Marrow Stem Cell Transplantation; Brain; C57BL/6 Mouse; CD34 gene; Cell Death; Cells; Childhood; Chronic; Clinical; Clinical Trials; Colony-Forming Units Assay; Commit; Cysteamine; Cystine; Cystinosis; Defect; Diabetes Mellitus; Disease; Disease Progression; Dose; Electrolytes; End stage renal failure; Engraftment; Eye; Eyedrops; Family; Fanconi Syndrome; Functional disorder; Gene Delivery; Gene-Modified; Genes; Graft Rejection; Growth; Hematopoietic; Hematopoietic stem cells; Hereditary Disease; Hour; Human; Hypogonadism; Hypothyroidism; Immunity; Immunosuppression; In Vitro; Individual; Injury; Insertional Mutagenesis; Isogenic transplantation; Kidney; Kidney Transplantation; Lentivirus Vector; Life; Liquid substance; Liver; Lymphoid Cell; Measures; Metabolic; Metabolic Diseases; Mus; Muscle; Myeloid Cells; Myopathy; Neuraxis; Odors; Organ; Patients; Peripheral Blood Stem Cell; Pharmaceutical Preparations; Pharmacology and Toxicology; Phase; Phase I Clinical Trials; Photophobia; Population; Proteins; Renal function; Research Personnel; Rickets; Risk; Safety; Site; Source; Stem cell transplant; Stem cells; Symptoms; Testing; Tissue Sample; Tissues; Toxic effect; Toxicology; Transplantation; Viral Vector; Vomiting; Work; cellular transduction; efficacy testing; gene therapy; genotoxicity; in vitro Assay; in vivo; infancy; injured; leukemic stem cell; mortality; mouse model; neuronal cell body; pre-clinical; preclinical study; prevent; progenitor; public health relevance; safety study; stem; stem cell therapy; treatment strategy; vector

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.

描述（申请人提供）：胱氨酸病是一种以细胞内胱氨酸积累为特征的代谢性遗传性疾病。受影响的个体通常会出现 一岁前出现近端肾小管病变，最终进展为终末期肾功能衰竭。胱氨酸积累导致多器官功能障碍。半胱胺药物可降低细胞内胱氨酸含量。然而，半胱胺不能预防近端肾小管病变，也不能预防终末期肾衰竭，只能延缓疾病的进展。该项目的长期目标是通过移植经过离体基因改造表达功能性 CTNS 基因的自体造血干细胞和祖细胞 (HSPC) 来开发治疗胱氨酸病的新疗法。使用胱氨酸中毒小鼠模型（Ctns-/- 小鼠），我们发现移植表达 Ctns 的同基因 Sca1+ HSPC 会导致丰富的骨髓来源细胞植入，并且所有测试组织中胱氨酸含量显着降低。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体 pCCL-CTNS 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，即该策略可以在疾病显着进展之前对患有胱氨酸中毒的年轻患者起作用。在获得 FDA 批准进行预研究者新药 (IND) 申请后，我们现在提议进行获得 IND 所需的药理学/毒理学研究，用于胱氨酸中毒的 I 期临床试验。这种治疗将代表一种终生治疗，可以预防肾移植和与胱氨酸中毒相关的长期并发症。慢病毒载体已证明其在小鼠和人类中长期转导 HSPC 的功效。所有药理学/毒理学研究都将使用根据良好生产规范生产的临床前批次的载体 pCCL-CTNS 进行。在具体目标 1 中，我们建议使用我们的载体 pCCL-CTNS 优化人类 CD34+ 细胞的转导，并使用集落形成单位 (CFU) 测定测试转导细胞通过谱系定向祖细胞而不变成白血病的能力。另一种评估整合病毒载体遗传毒性的体外测定是体外永生化 (IVIM) 测定，使用 鼠谱系阴性骨髓细胞。载体拷贝数 (VCN) 和载体整合位点 (VIS) 将在两个测定的最终集落和克隆中确定。在具体目标 2 中，我们将使用由 pCCL-CTNS 离体转导的鼠 Sca1+ HSPC 并移植到初级和次级受体 Ctns-/- 小鼠中，在体内测试该策略的有效性和安全性。疗效将通过血液和组织样本中的 CTNS 表达、组织胱氨酸水平和肾功能来衡量。毒性将通过综合临床和组织学组织分析、评估骨髓和淋巴细胞中的 VCN 和 VIS 以及检测对 CTNS 蛋白的潜在抗体免疫力来确定。这项工作代表了胱氨酸沉积症 I 期临床试验的最后一步，也是治疗其他溶酶体贮积症的概念证明。