Macrophage-based Human Gene Therapy for Hereditary PAP

基于巨噬细胞的遗传性 PAP 人类基因治疗

• 批准号: 8206634
• 负责人: Bruce C Trapnell
• 金额: $ 21.88万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-15 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8206634
• 关键词: Address; Adult; Affect; Alternative Therapies; Alveolar Macrophages; Alveolus; Animals; Autoantibodies; Autoimmune Process; Autologous; Autologous Bone Marrow Transplantation; Bacteriophages; Biochemistry; Biological Assay; Biological Markers; Bone Marrow; Bone Marrow Purging; Bone Marrow Transplantation; Breathing; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Bronchoscopes; CD34 gene; CSF2RA gene; CSF2RB gene; CSF3 gene; Catabolism; Cell Therapy; Cells; Child; Childhood; Cyclophosphamide; Cytology; Data; Diagnosis; Disease; Engraftment; Figs - dietary; Flow Cytometry; Functional disorder; Future; General Anesthesia; Genes; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor Receptors; Growth Factor; Hereditary Disease; Histologic; Histopathology; Human; ITGAM gene; Immune response; Implant; Individual; Infection; Inherited; Lentivirus Vector; Lobe; Lung; Lung diseases; Measures; Mechanics; Mediating; Methods; Molecular Diagnosis; Mus; Mutation; Myeloid Cells; Pathogenesis; Patients; Phosphorylation; Procedures; Proteins; Pulmonary Alveolar Proteinosis; Pulmonary Surfactants; Receptor Gene; Reporting; Respiratory Failure; Respiratory Insufficiency; Retroviridae; Risk; STAT5A gene; Saline; Serum; Signal Transduction; Sodium Chloride; Testing; Therapeutic; Transduction Gene; Unconscious State; Virus; Water; base; cellular transduction; cohort; effective therapy; functional restoration; gene correction; gene therapy; gene therapy clinical trial; improved; in vivo; macrophage; molecular pathology; monocyte; mouse model; nonhuman primate; novel; novel strategies; peripheral blood; pre-clinical; receptor; receptor expression; receptor function; response; restoration; rituximab; success; surfactant; uptake

ABSTRACT Recessive mutations in CSF2RA, the gene encoding the ¿-chain of the granulocyte/macrophage-colony stimulating factor receptor (GM-R), were recently reported by the applicant as a newly-identified genetic disorder causing pulmonary alveolar proteinosis (PAP) in children. Recognizing increased serum GM-CSF as a disease biomarker, the applicant identified a cohort of affected individuals and defined the pathogenesis, presentation, diagnosis, molecular pathophysiology, and therapy response of hereditary PAP. The disease is characterized by the progressive accumulation of surfactant in alveolar macrophages (AM) and alveoli, resulting in respiratory insufficiency and, in severe cases, respiratory failure. Current therapy is whole lung lavage, a procedure performed under general anesthesia in which one lung is mechanical ventilated while the other is repeatedly filled with warmed saline, mechanically percussed to emulsify the accumulated surfactant, and then drained to physically remove it. The procedure is repeated as required, which in some children is every two months. PAP occurs in genetically modified mice deficient in the ¿-chain of the GM-R (GM-R¿KO mice) or in GM-CSF (GMKO mice), and in humans with neutralizing GM-CSF autoantibodies (autoimmune PAP), all of which result in pulmonary histopathology and molecular pathology similar to hereditary PAP in children. Without stimulation by GM-CSF, surfactant catabolism in AM is impaired and results in reduced pulmonary clearance and progressive accumulation of pulmonary surfactant. Bone marrow transplantation (BMT) is a therapeutic option for these children with PAP and was attempted in one child who died of a lung infection before engraftment was complete. The accumulated surfactant in PAP likely increases the infection risk associated with myeloablation, which is required for BMT. PAP in GM-R¿KO mice was 'cured' by the applicant by autologous transplantation of bone marrow after retrovirus-mediated gene transduction to correct GM-R function. The central hypothesis of this proposal is that ex-vivo lentiviral vector-mediated restoration of functional GM-R expression in autologous monocytes or G-CSF-mobilized CD34+ cells followed by intrapulmonary administration into non-myeloablated recipients will be safe, well-tolerated, and effective therapy of hPAP. This hypothesis will be tested in 3 Specific Aims: (1) macrophage-mediated cell therapy of hPAP in mice; (2) macrophage-mediated gene therapy of hPAP in mice; (3) preclinical correction of CSF2RA expression and surfactant catabolism in macrophages from children with hPAP, evaluated in vivo in the lungs of non-human primates. A novel approach avoiding known impediments to lung gene therapy will take advantage of the natural survival advantage of GM-R gene-corrected cells conferred by the elevated levels of GM-CSF, a potent growth factor for both human and murine AM. This approach gives hereditary PAP an outstanding chance of being the first human lung disease to be successfully treated by gene therapy and provides a feasible and potential therapeutic alternative for a devastating disease in children.

摘要 粒细胞/巨噬细胞集落<$-链编码基因CSF 2 RA的隐性突变 刺激因子受体（GM-R），最近被申请人报道为一种新鉴定的遗传因子， 导致儿童肺泡蛋白沉积症（PAP）的疾病。认识到血清GM-CSF升高是一种 疾病生物标志物，申请人鉴定了一组受影响的个体并定义了发病机制， 遗传性PAP的表现、诊断、分子病理生理学和治疗反应。述疾病是 其特征在于肺泡巨噬细胞（AM）和肺泡中表面活性剂的进行性积累， 导致呼吸功能不全，严重的情况下导致呼吸衰竭。目前的治疗是全肺 灌洗，一种在全身麻醉下进行的手术，其中一个肺机械通气， 另一个是重复地用温热的盐水填充，机械搅拌以除去积聚的表面活性剂， 然后排干以物理去除它。该过程根据需要重复，在一些儿童中， 每两个月一次PAP发生在GM-R <$-链（GM-R <$KO）缺陷的转基因小鼠中 小鼠）或GM-CSF（GMKO小鼠）中，以及在具有中和GM-CSF自身抗体（自身免疫性 PAP），所有这些导致肺组织病理学和分子病理学类似于遗传性PAP， 孩子在没有GM-CSF刺激的情况下，AM中的表面活性剂催化剂受损并导致降低的细胞增殖。 肺清除率和肺表面活性物质的进行性蓄积。骨髓移植 (BMT)是这些PAP儿童的治疗选择，并在一名死于肺部疾病的儿童中进行了尝试 植入前感染完成。PAP中积聚的表面活性剂可能增加感染 与骨髓消融相关的风险，这是BMT所需的。GM-R基因敲除小鼠的PAP被 申请人通过自体骨髓移植后逆转录病毒介导的基因转导来纠正 GM-R功能。这一提议的中心假设是，离体慢病毒载体介导的细胞凋亡的恢复， 在自体单核细胞或G-CSF动员的CD 34+细胞中的功能性GM-R表达， 肺内给药到非骨髓清除的受者将是安全的、耐受良好的和有效的 hPAP的治疗这一假设将在3个具体目标中进行检验：（1）巨噬细胞介导的细胞疗法， 小鼠hPAP;（2）巨噬细胞介导的小鼠hPAP基因治疗;（3）CSF 2 RA的临床前校正 hPAP患儿巨噬细胞中的表达和表面活性剂催化剂，在肺中进行体内评价 非人类灵长类动物。一种新的方法，避免已知的障碍，肺基因治疗将采取 GM-R基因校正的细胞的天然存活优势的优势，其由升高的GM-R基因水平赋予。 GM-CSF，一种对人和鼠AM都有效的生长因子。这种方法使遗传性PAP 成为第一个通过基因治疗成功治疗的人类肺部疾病的绝佳机会， 为儿童的毁灭性疾病提供了一种可行和潜在的治疗选择。