Macrophage-based Human Gene Therapy for Hereditary PAP

描述（由申请人提供）：CSF2RA的隐性突变是编码粒细胞/巨噬细胞集落刺激因子受体（GM-R） 1链的基因，最近被申请人报道为一种新发现的导致儿童肺泡蛋白沉积症（PAP）的遗传疾病。认识到血清GM-CSF升高是一种疾病生物标志物，申请人确定了一组受影响的个体，并确定了遗传性PAP的发病机制、表现、诊断、分子病理生理和治疗反应。该疾病的特征是表面活性剂在肺泡巨噬细胞（AM）和肺泡中逐渐积累，导致呼吸功能不全，严重时可导致呼吸衰竭。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，其中一个肺是机械通气的，而另一个肺反复充满温盐水，机械敲击以乳化积累的表面活性剂，然后排干以物理去除它。这一过程可根据需要重复进行，有些儿童每两个月进行一次。PAP发生在缺乏GM-R -链的转基因小鼠（GM-R - KO小鼠）或GM-CSF （GMKO小鼠）中，以及具有中和GM-CSF自身抗体（自身免疫性PAP）的人类中，所有这些都导致肺部组织病理学和分子病理学类似于儿童遗传性PAP。如果没有GM-CSF的刺激，AM中的表面活性剂分解代谢受损，导致肺清除率降低和肺表面活性剂的进行性积累。骨髓移植（BMT）是这些患有PAP的儿童的一种治疗选择，并在一名在植入完成前死于肺部感染的儿童中进行了尝试。PAP中积累的表面活性剂可能会增加骨髓消融相关的感染风险，而骨髓消融是治疗BMT所必需的。在逆转录病毒介导的基因转导纠正GM-R功能后，申请人通过自体骨髓移植“治愈”了GM-R2KO小鼠的PAP。该建议的中心假设是，体外慢病毒载体介导的在自体单核细胞或g - csf动员的CD34+细胞中恢复功能性GM-R表达，然后肺内给药到非清髓受体中，将是安全、耐受性良好且有效的hPAP治疗方法。这一假设将在3个特定目标中得到验证：(1)巨噬细胞介导的小鼠hPAP细胞治疗；(2)巨噬细胞介导的小鼠hPAP基因治疗；(3)临床前校正hPAP患儿巨噬细胞中CSF2RA表达和表面活性剂分解代谢，并在非人类灵长类动物肺中进行体内评估。一种避免肺部基因治疗已知障碍的新方法将利用GM-R基因校正细胞的天然生存优势，这种优势是由人类和小鼠AM的一种有效生长因子GM-CSF水平升高所赋予的。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，并为儿童毁灭性疾病提供了可行和潜在的治疗选择。

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.