Macrophage-based Human Gene Therapy for Hereditary PAP

摘要 编码粒细胞/巨噬细胞集落链基因CSF2RA的隐性突变 刺激因子受体(GM-R)是最近被申请人报道的一种新发现的基因 引起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 疾病生物标记物，申请人确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。这种疾病是 以肺泡巨噬细胞(AM)和肺泡内表面活性物质的进行性积聚为特征， 导致呼吸功能不全，在严重情况下，还会导致呼吸衰竭。目前的治疗方法是全肺 灌洗，在全身麻醉下进行的一种手术，其中一只肺被机械通风，而另一只肺 另一种是反复填充温盐水，机械冲击以乳化积累的表面活性物质， 然后排干，以便将其物理移除。根据需要重复这一过程，在一些儿童中是这样 每两个月一次。PAP发生在GM-R(GM-R？KO)链缺陷的转基因小鼠身上 小鼠)或GM-CSF(GMKO小鼠)，以及具有中和GM-CSF自身抗体的人类(自身免疫 PAP)，所有这些都导致与遗传性PAP相似的肺组织病理学和分子病理学。 孩子们。在没有GM-CSF刺激的情况下，AM中的表面活性物质分解代谢受到损害，导致 肺清除和肺表面活性物质进行性蓄积。骨髓移植 (BMT)是这些患有PAP的儿童的一种治疗选择，曾尝试在一名死于肺的儿童身上进行 植入术完成前发生感染。PAP中积累的表面活性物质可能会增加感染 骨髓移植所需的骨髓清除术相关风险。GM-R？KO小鼠中的PAP可通过 逆转录病毒介导基因转导后自体骨髓移植矫正 GM-R功能。这一建议的中心假设是体外慢病毒载体介导的修复 GM-R在自体单核细胞或G-CSF动员的CD34+细胞中的功能性表达 对非清髓受者进行肺内给药将是安全、耐受性好和有效的 HPAP的治疗。这一假设将在三个具体目标中得到验证：(1)巨噬细胞介导的细胞治疗 (2)巨噬细胞介导的小鼠HPAP基因治疗；(3)CSF2RA的临床前纠正 HPAP患儿肺内巨噬细胞表达和表面活性物质分解代谢的活体研究 非人灵长类动物。一种避免肺部基因治疗已知障碍的新方法将采取 GM-R基因修饰细胞的自然存活优势是由于 GM-CSF--人和小鼠AM的有效生长因子这种方法为遗传性PAP提供了 极有可能成为第一个成功通过基因治疗和治疗的人类肺部疾病 为儿童的一种毁灭性疾病提供了一种可行且潜在的治疗选择。

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.