Macrophage-based Human Gene Therapy for Hereditary PAP

描述（申请人提供）：申请人最近报告了CSF 2 RA（编码粒细胞/巨噬细胞集落刺激因子受体（GM-R）1-链的基因）的隐性突变，作为一种新发现的导致儿童肺泡蛋白沉积症（PAP）的遗传性疾病。申请人认识到血清GM-CSF升高是一种疾病生物标志物，确定了一组受影响的个体，并定义了遗传性PAP的发病机制、表现、诊断、分子病理生理学和治疗反应。该疾病的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，并且在严重的情况下，导致呼吸衰竭。目前的治疗方法是全肺灌洗，这是一种在全身麻醉下进行的手术，其中一个肺机械通气，而另一个肺反复填充温盐水，机械冲洗以去除积聚的表面活性剂，然后排出以物理去除它。该手术根据需要重复进行，在一些儿童中每两个月进行一次。PAP发生在GM-R <$链缺陷的转基因小鼠（GM-R <$KO小鼠）或GM-CSF缺陷的转基因小鼠（GMKO小鼠）中，以及具有中和GM-CSF自身抗体（自身免疫PAP）的人类中，所有这些都导致与儿童遗传性PAP相似的肺组织病理学和分子病理学。在没有GM-CSF刺激的情况下，AM中的表面活性物质催化剂受损，并导致肺清除率降低和肺表面活性物质的进行性积聚。骨髓移植（BMT）是这些PAP儿童的治疗选择，并尝试在一个孩子谁死于肺部感染前植入完成。PAP中积累的表面活性剂可能增加骨髓消融相关的感染风险，这是BMT所需的。在逆转录病毒介导的基因转导以纠正GM-R功能后，申请人通过自体骨髓移植“治愈”了GM-R2 KO小鼠中的PAP。该建议的中心假设是，在自体单核细胞或G-CSF动员的CD 34+细胞中，体外慢病毒载体介导的功能性GM-R表达的恢复，然后肺内给药至非骨髓清除的受体，将是安全的、耐受性良好的和有效的hPAP治疗。将在3个特定目的中检验该假设：（1）小鼠中巨噬细胞介导的hPAP细胞疗法;（2）小鼠中巨噬细胞介导的hPAP基因疗法;（3）在非人灵长类动物肺中体内评价的来自hPAP儿童的巨噬细胞中CSF 2 RA表达和表面活性剂catalysts的临床前校正。一种避免已知肺基因治疗障碍的新方法将利用GM-CSF水平升高所赋予的GM-R基因校正细胞的自然存活优势，GM-CSF是人类和小鼠AM的有效生长因子。这种方法使遗传性PAP成为第一种通过基因治疗成功治疗的人类肺部疾病，并为儿童的毁灭性疾病提供了可行和潜在的治疗选择。 公共卫生相关性：申请人最近在儿童中发现了一种新的肺部疾病（遗传性PAP），该疾病是由肺正常功能所需的天然油性物质（表面活性剂）蓄积引起的。这种疾病是由一种基因突变引起的，这种基因的蛋白产物（GM-CSF受体）是肺细胞（肺泡巨噬细胞）清除表面活性剂所必需的。目前的治疗方法包括在患者无意识和用机器呼吸的情况下，用大量盐水反复洗涤肺部，以物理方式去除表面活性剂。拟议的研究将在该疾病的小鼠模型中测试一种新的基因治疗方法，并通过病毒介导的正常基因校正，然后注入非人类灵长类动物的肺部，来校正受影响儿童的缺陷细胞。预期的临床前结果需要监管机构批准的未来临床试验的基因治疗的遗传性PAP的儿童。

DESCRIPTION (provided by applicant): Recessive mutations in CSF2RA, the gene encoding the 1-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-R2KO 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. PUBLIC HEALTH RELEVANCE: The applicant recently identified a new lung disease (hereditary PAP) in children caused by accumulation of the natural oily substance (surfactant) required for the lungs to function normally. The disease is caused by mutations in a gene whose protein product (GM-CSF receptor) is required by lung cells (alveolar macrophages) to remove surfactant. Current therapy involves repeatedly washing the lungs with large volumes of salt water to physically remove surfactant while the patient is unconscious and breathing with a machine. The proposed studies will test a new method of gene therapy in a mouse model of the disease and also correction of defective cells from affected children by virus-mediated correction with a normal gene followed by instillation into the lungs of non-human primates. Anticipated preclinical results are needed for regulatory approval for a future clinical trial of gene therapy of hereditary PAP in children.