Macrophage Based Gene Therapy for Hereditary Pulmonary Alveolar Proteinosis

描述(申请人提供)：遗传性肺泡蛋白沉积症(HPAP)是一种表面活性物质积聚增加导致呼吸衰竭的疾病，目前尚无药物治疗。它是由于编码GM-CSF受体(GM-R)α和β亚基的CSF2RA或CSF2RB突变而导致的肺泡巨噬细胞(AM)的GM-CSF信号中断所致。其长期目标是开发基因疗法来恢复AM中的GM-CSF信号，从而恢复AM对表面活性物质清除、肺泡内环境稳定、肺功能和宿主防御至关重要的GM-CSF依赖的AM功能。本文的目的是评价一种新的治疗方法-肺巨噬细胞移植(PMT)。初步数据显示，GM-R缺陷(GM-RKO)小鼠患上与人类HPAP相同的HPAP肺部疾病，包括肺GM-CSF水平升高，这为具有GM-R功能的AM提供了选择性生存优势。中心假设是，安全增强的慢病毒载体介导的GM-R在造血干细胞/前体细胞(HSPC)中的表达，扩增到巨噬细胞，以及不经骨髓清髓的基因校正细胞的自体PMT将是有效和安全的HPAP治疗。初步数据显示，一次PMT治疗可以纠正GM-RKO小鼠至少一年的HPAP(评估的最长时间)，没有相关的不良事件，并且可以很容易地使用申请人实验室中成熟的方法转导人HSPC，并将其扩增为表达功能性GM-R的巨噬细胞。理由是预期结果将为未来临床试验的设计提供信息，并提供临床前安全性和有效性数据，以及GMP 在人体上测试这种方法所需的制造程序和验证数据，以获得监管部门的批准。该假说将从四个具体目标进行验证：1)确定HPAP在GM-RKO小鼠中的基因转移/PMT治疗的效率和动力学；2)优化具有最大PMT植入潜力的人HSPC来源的、经过基因修正的巨噬细胞的扩增；3)在与基因转移、HSPC来源的巨噬细胞扩增和GM-RKO小鼠中转导细胞的药理耗竭相关的临床前研究中，确定基因转移/PMT的安全性；4)开发和验证生产HSPC来源的、经过基因修正的巨噬细胞的方案，并为HPAP的基因转移/PMT治疗制定临床方案和研究新药应用。这种方法是创新的，因为它明显不同于目前低效、高侵入性的全肺灌洗物理清除表面活性物质的方法，而是使用了一种新的方法来恢复AM功能。这项拟议的研究具有重要意义，因为它有望建立一种针对HPAP儿童的特定疗法和一种可能对其他疾病有用的新型疗法(PMT)的可行性，并评估多种安全性改进以降低基因疗法的风险。这些结果将揭示GM-CSF调节AM群体大小的基本机制，提供AM寿命的估计，并为巨噬细胞治疗的发展奠定基础。

DESCRIPTION (provided by applicant): Hereditary pulmonary alveolar proteinosis (hPAP) is a disorder of increased surfactant accumulation resulting in respiratory failure for which no pharmacologic therapy exists. It is caused by disruption of GM-CSF signaling to alveolar macrophages (AMs) by mutations in CSF2RA or CSF2RB, which encode GM-CSF receptor (GM-R) α and β subunits, respectively. The long-term goal is to develop gene therapy to restore GM-CSF signaling in AMs and, thereby, GM-CSF-dependent AM functions critical to surfactant clearance, alveolar homeostasis, lung function and host defense. The objective here is to evaluate a novel therapeutic approach, pulmonary macrophage transplantation (PMT). Preliminary data show that GM-R deficient (GM-RKO) mice develop hPAP lung disease that is identical to hPAP in humans including an increased level of pulmonary GM-CSF, which confers a selective survival advantage to AMs with functional GM-Rs. The central hypothesis is that safety-enhanced, lentiviral vector-mediated GM-R expression in hematopoietic stem/precursor cells (HSPCs), expansion into macrophages, and autologous PMT of gene-corrected cells without myeloablation will be effective and safe as therapy of hPAP. Preliminary data show that a single PMT treatment can correct hPAP in GM-RKO mice for at least one year (the longest time evaluated) without associated adverse events, and that human HSPCs can be readily transduced using well-established methods in the applicants' laboratory and expanded into macrophages expressing functional GM-Rs. The rationale is that anticipated results will inform the design of future a clinical trial and provide the preclinical safety and efficacy data, and GMP manufacturing procedures and validation data required to obtain regulatory approval to test this approach in humans. The hypothesis will be tested in four specific aims: 1) determine the efficiency and kinetics of gene transfer/PMT therapy of hPAP in GM-RKO mice; 2) optimize the expansion of human HSPC-derived, gene- corrected macrophages with maximum PMT engraftment potential; 3) determine the safety of gene transfer/PMT in preclinical studies related to gene transfer, macrophage expansion from HSPCs, PMT, and pharmacologic depletion of transduced cells in GM-RKO mice; 4) develop and validate protocols for the manufacture of HSPC-derived, gene-corrected macrophages, and write a clinical protocol and investigational new drug application for gene transfer/PMT therapy of hPAP. The approach is innovative because it departs markedly from the current inefficient, highly invasive method of physically removing surfactant by whole lung lavage and in- stead uses a novel approach to restore AM function. The proposed research is significant because it is expected to establish the feasibility of a specific therapy for children with hPAP and a new type of therapy (PMT) that may be useful for other diseases, and evaluate multiple safety improvements to reduce the risks of gene therapy. Results will inform a fundamental mechanism by which GM-CSF regulates AM population size, pro- vide an estimate of AM lifespan, and lay the foundation for the development of macrophage-based therapy.