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; 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（由申请人提供）：膀胱变性是一种代谢遗传疾病，其特征是细胞内积累。受影响的个体通常与 一岁之前的近端微管病，并最终发展为末期肾衰竭。胱氨酸积累会导致多器官功能障碍。药物赛梯减少细胞内胱氨酸含量。然而，cysteamine并不能阻止近端微型肾上腺病或末期肾衰竭，而只会延迟疾病的进展。该项目的长期目标是通过移植自体造血干细胞和祖细胞（HSPC）基因改性的离体来开发新的囊肿性治疗方法，以表达功能性CTNS基因。使用小鼠模型进行囊肿性，CTNS - / - 小鼠，我们表明表达CTN的同性SCA1+ HSPC的移植导致大量的骨髓衍生的细胞植入和大量减少所测试的组织中的胱氨酸含量。这种治疗也阻止了肾功能障碍的进展。我们使用慢病毒载体构建体PCCL-CTN获得了离体转导的HSPC相同的结果，并确定了CTNS-/ - 小鼠中的首次概念证明，该策略可以在年轻患者的伴有囊肿性疾病之前的年轻患者起作用。在获得FDA的批准以继续进行研究前的新药（IND）应用后，我们现在提出了为获得I期cystinosis的I阶段IDS所需的药理学/毒理学研究。这种治疗方法将代表终身，可以防止肾脏移植和与囊肿性相关的长期并发症。慢病毒载体证明了其对小鼠和人类的长期HSPC转导的功效。所有的药理学/毒理学研究都将通过在良好的制造实践下产生的临床前批次PCCL-CTN进行。在特定的目标1中，我们建议使用我们的载体PCCL-CTN优化人CD34+细胞的转导，并测试转导细胞通过谱系共同的祖细胞的能力，而无需使用菌落形成单位（CFU）测定。评估整合病毒载体遗传毒性的另一个体外测定是使用体外永生化（IVIM）测定法 鼠谱系阴性骨髓细胞。向量拷贝数（VCN）和向量集成位点（VIS）将在最终菌落和克隆中确定这两个测定法。在特定的目标2中，我们将使用PCCL-CTN转导并移植在初级和次要接受者CTNS的鼠 - / - 小鼠中，使用鼠SCA1+ HSPC在体内测试该策略的功效和安全性。功效将通过CTNS表达在血液和组织样品，组织胱氨酸水平和肾功能中来衡量。毒性将由全面的临床和组织学组织分析确定，通过评估髓样和淋巴样细胞中的VCN和VIS并检测对CTNS蛋白的潜在抗体免疫。这项工作代表了朝着炎症的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证明。