Hereditary Pulmonary Alveolar Proteinosis Research Articles

A toxicology study of Csf2ra complementation and pulmonary macrophage transplantation therapy of hereditary PAP in mice

Pulmonary macrophage transplantation (PMT) is a gene and cell transplantation approach in development as therapy for hereditary pulmonary alveolar proteinosis (hPAP), a surfactant accumulation disorder caused by mutations in CSF2RA/B (and murine homologs). We conducted a toxicology study of PMT of Csf2ra gene-corrected macrophages (mGM-Rα+Mϕs) or saline-control intervention in Csf2raKO or wild-type (WT) mice including single ascending dose andrepeat ascending dose studies evaluating safety, tolerability, pharmacokinetics, and pharmacodynamics. Lentiviral-mediated Csf2ra cDNA transfer restored GM-CSF signaling in mGM-Rα+Mϕs. Following PMT, mGM-Rα+Mϕs engrafted, remained within the lungs, and did not undergo uncontrolled proliferation or result in bronchospasm, pulmonary function abnormalities, pulmonary or systemic inflammation, anti-transgene product antibodies, or pulmonary fibrosis. Aggressive male fighting caused a similarly low rate of serious adverse events in saline- and PMT-treated mice. Transient, minor pulmonary neutrophilia and exacerbation of pre-existing hPAP-related lymphocytosis were observed 14days after PMT of the safety margin dose but not the target dose (5,000,000 or 500,000 mGM-Rα+Mϕs, respectively) and only in Csf2raKO mice but not in WT mice. PMT reduced lung disease severity in Csf2raKO mice. Results indicate PMT of mGM-Rα+Mϕs was safe, well tolerated, and therapeutically efficacious in Csf2raKO mice, andestablished a no adverse effect level and 10-fold safety margin.

Hereditary Pulmonary Alveolar Proteinosis in a Pediatric Patient – A Diagnostic and Therapeutic Challenge

Abstract The abstract will be: “Pulmonary Alveolar Proteinosis (PAP) is a rare but potentially fatal respiratory disorder. The hereditary form is due to mutations affecting the GM-CSF receptor subunits. We are presenting this case report of a 3-year-old girl -who was the child of a fourth-degree consanguineous marriage- who developed Hereditary Alveolar Proteinosis. Her diagnosis was confirmed by surgical biopsy as well as whole exome sequencing (WES) genetic testing that revealed homozygous gene deletion of the CSF2RA gene (Colony Stimulating Factor 2RA). She had a favorable outcome and spontaneous resolution of her disease on follow-up.”

CSF2RB mutation-related hereditary pulmonary alveolar proteinosis: the "long and winding road" into adulthood.

Genetic analysis pre-lung transplantation diagnosed a case of hereditary pulmonary alveolar proteinosis (PAP) complicated by fibrosis in adulthood. The need for genetic testing in GM-CSF autoantibody negative and unclassifiable PAP is highlighted. https://bit.ly/3QcsYwM.

Research progress of cell therapy in hereditary pulmonary alveolar proteinosis

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare interstitial lung disease caused by mutation in CSF2RA/CSF2RB, characterized by the deposition of pulmonary surfactant due to the alveolar macrophage dysfunction. The whole lung lavage can effectively alleviate the symptoms but is associated with potential complications. Cell therapy is a novel approach with advances that provide a new therapeutic strategy for the treatment of hPAP.

A murine model of hereditary pulmonary alveolar proteinosis caused by homozygous Csf2ra gene disruption.

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder caused by recessive mutations in GM-CSF receptor subunit α/β genes (CSF2RA/CSF2RB, respectively) characterized by impaired GM-CSF-dependent surfactant clearance by alveolar macrophages (AMs) resulting in alveolar surfactant accumulation and hypoxemic respiratory failure. Because hPAP is caused by CSF2RA mutations in most patients, we created an animal model of hPAP caused by Csf2ra gene disruption (Csf2ra-/- mice) and evaluated the effects on AMs and lungs. Macrophages from Csf2ra-/- mice were unable to bind and clear GM-CSF, did not exhibit GM-CSF signaling, and had functional defects in phagocytosis, cholesterol clearance, and surfactant clearance. Csf2ra-/- mice developed a time-dependent, progressive lung disease similar to hPAP in children caused by CSF2RA mutations with respect to the clinical, physiological, histopathological, biochemical abnormalities, biomarkers of PAP lung disease, and clinical course. In contrast, Csf2ra+/- mice had functionally normal AMs and no lung disease. Pulmonary macrophage transplantation (PMT) without myeloablation resulted in long-term engraftment, restoration of GM-CSF responsiveness to AMs, and a safe and durable treatment effect that lasted for the duration of the experiment (6 mo). Results demonstrate that homozygous (but not heterozygous) Csf2ra gene ablation caused hPAP identical to hPAP in children with CSF2RA mutations, identified AMs as the cellular site of hPAP pathogenesis in Csf2ra-/- mice, and have implications for preclinical studies supporting the translation of PMT as therapy of hPAP in humans.

Hereditary pulmonary alveolar proteinosis as collateral damage from a large chromosomal deletion.

A girl with a known chromosomal deletion at Xp22.33, learning difficulties and short stature presented with dyspnea and dry cough and an abnormal chest X-ray. Computed tomography was typical for pulmonary alveolar proteinosis (PAP), and the diagnosis was confirmed invasively. More detailed genetic analysis detected a homozygous deletion of the colony-stimulating factor-2-receptor alpha subunit (CSF2RA) gene. In this patient, the Xp22.33 deletion affected 8 genes, including CSF2RA, leading to GM-CSF receptor dysfunction and hereditary PAP. This is the first report of childhood interstitial lung disease (chILD) as collateral damage from a large chromosomal deletion.

Genetic determinants of risk in autoimmune pulmonary alveolar proteinosis

Pulmonary alveolar proteinosis (PAP) is a devastating lung disease caused by abnormal surfactant homeostasis, with a prevalence of 6–7 cases per million population worldwide. While mutations causing hereditary PAP have been reported, the genetic basis contributing to autoimmune PAP (aPAP) has not been thoroughly investigated. Here, we conducted a genome-wide association study of aPAP in 198 patients and 395 control participants of Japanese ancestry. The common genetic variant, rs138024423 at 6p21, in the major-histocompatibility-complex (MHC) region was significantly associated with disease risk (Odds ratio [OR] = 5.2; P = 2.4 × 10−12). HLA fine-mapping revealed that the common HLA class II allele, HLA-DRB1*08:03, strongly drove this signal (OR = 4.8; P = 4.8 × 10−12), followed by an additional independent risk allele at HLA-DPβ1 amino acid position 8 (OR = 0.28; P = 3.4 × 10−7). HLA-DRB1*08:03 was also associated with an increased level of anti-GM-CSF antibody, a key driver of the disease (β = 0.32; P = 0.035). Our study demonstrated a heritable component of aPAP, suggesting an underlying genetic predisposition toward an abnormal antibody production.

Shaping the future of an ultra-rare disease: unmet needs in the diagnosis and treatment of pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) can be considered the archetype of ultra-rare diseases with a prevalence of under 10 cases per million. We discuss the classification of PAP, the current diagnostic practice and the supplementary role of genetic testing and granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in the diagnosis of congenital and hereditary PAP. We report on novel therapeutic approaches such as GM-CSF substitution, stem cell transplantation, pioglitazone, statins and immunomodulation. The discovery of new genetic mutations underlying this syndrome raises the question whether the classification should be radically revised in the future. Serum GM-CSF autoantibody is the best diagnostic marker for autoimmune PAP, the most common form, but does not correlate with disease severity. Several circulating biomarkers have been investigated to assess disease activity and predict outcome. Imaging techniques have also enormously evolved and offer new tools to quantify disease burden and possibly drive therapeutic decisions. Promising clinical trials are ongoing and will generate new treatment strategies besides or in addition to whole lung lavage in the next future. Despite impressive advances in understanding pathogenesis, PAP remains a rare syndrome with several unanswered questions impacting diagnosis, management and treatment, and, as a result, patients' quality of life.

Effective hematopoietic stem cell-based gene therapy in a murine model of hereditary pulmonary alveolar proteinosis

Hereditary pulmonary alveolar proteinosis due to GM-CSF receptor deficiency (herPAP) constitutes a life-threatening lung disease characterized by alveolar deposition of surfactant protein secondary to defective alveolar macrophage function. As current therapeutic options are primarily symptomatic, we have explored the potential of hematopoietic stem cell-based gene therapy. Using Csf2rb−/− mice, a model closely reflecting the human herPAP disease phenotype, we here demonstrate robust pulmonary engraftment of an alveolar macrophage population following intravenous transplantation of lentivirally corrected hematopoietic stem and progenitor cells. Engraftment was associated with marked improvement of critical herPAP disease parameters, including bronchoalveolar fluid protein, cholesterol and cytokine levels, pulmonary density on computed tomography scans, pulmonary deposition of Periodic Acid-Schiff+ material as well as respiratory mechanics. These effects were stable for at least nine months. With respect to engraftment and alveolar macrophage differentiation kinetics, we demonstrate the rapid development of CD11c+/SiglecF+ cells in the lungs from a CD11c–/SiglecF+ progenitor population within four weeks after transplantation. Based on these data, we suggest hematopoietic stem cell-based gene therapy as an effective and cause-directed treatment approach for herPAP.

Long-Term Safety and Efficacy of Gene-Pulmonary Macrophage Transplantation Therapy of PAP in Csf2ra-/- Mice.

Hereditary pulmonary alveolar proteinosis (PAP) is a genetic lung disease characterized by surfactant accumulation and respiratory failure arising from disruption of GM-CSF signaling. While mutations in either CSF2RA or CSF2RB (encoding GM-CSF receptor α or β chains, respectively) can cause PAP, α chain mutations are responsible in most patients. Pulmonary macrophage transplantation (PMT) is a promising new cell therapy in development; however, no studies have evaluated this approach for hereditary PAP (hPAP) caused by Csf2ra mutations. Here, we report on the preclinical safety, tolerability, and efficacy of lentiviral-vector (LV)-mediated Csf2ra expression in macrophages and PMT of gene-corrected macrophages (gene-PMT therapy) in Csf2ra gene-ablated (Csf2ra-/-) mice. Gene-PMT therapy resulted in a stable transgene integrationand correction of GM-CSF signaling and functions in Csf2ra-/- macrophages invitro and invivo and resulted in engraftment and long-term persistence of gene-corrected macrophages in alveoli; restoration of pulmonary surfactant homeostasis; correction of PAP-specific cytologic, histologic, and biomarker abnormalities; and reduced inflammation associated with disease progression in untreated mice. No adverse consequences of gene-PMT therapy in Csf2ra-/- mice were observed. Results demonstrate that gene-PMT therapy of hPAP in Csf2ra-/- mice was highly efficacious, durable, safe, and well tolerated.

The influence of genetics on therapeutic developments in pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is characterized by the massive accumulation of lipoproteinaceous material within alveoli, which results in progressive respiratory failure. The abnormalities in surfactant clearance are caused by defective pulmonary macrophages, whose terminal differentiation is GM-CSF-dependent. In hereditary PAP, the rupture of GM-CSF signaling is because of mutations in the GM-CSF receptor genes. This review focus on the innovative technologies of gene-correction proposed for the development of new therapeutic strategies, for hereditary PAP patients. Hematopoietic stem cell gene therapy has been successfully experimented in murine models to restore the expression of the GM-CSF receptor, however, a therapeutic approach based on bone marrow transplantation requires a preconditioning, which could be hazardous in PAP patients, who are highly susceptible to pulmonary infections. Gene-corrected pulmonary macrophages, administered directly to the lung, could represent an improved approach. Finally, patient-derived induced pluripotent stem cells seem to be promising to overcome the limited availability of primary patient cells and to generate gene-corrected macrophages, able to recover pulmonary surfactant clearance. WLL is the gold standard therapy for PAP. However, its use in hereditary PAP is limited by the difficulty of performing this technique in paediatric patients and by its purely symptomatic efficacy. The recent advances in genome engineering could provide efficacious strategies for clinical application.

IPSC-Derived Macrophages Effectively Treat Pulmonary Alveolar Proteinosis in Csf2rb-Deficient Mice

SummaryInduced pluripotent stem cell (iPSC)-derived hematopoietic cells represent a highly attractive source for cell and gene therapy. Given the longevity, plasticity, and self-renewal potential of distinct macrophage subpopulations, iPSC-derived macrophages (iPSC-Mφ) appear of particular interest in this context. We here evaluated the airway residence, plasticity, and therapeutic efficacy of iPSC-Mφ in a murine model of hereditary pulmonary alveolar proteinosis (herPAP). We demonstrate that single pulmonary macrophage transplantation (PMT) of 2.5–4 × 106 iPSC-Mφ yields efficient airway residence with conversion of iPSC-Mφ to an alveolar macrophage (AMφ) phenotype characterized by a distinct surface marker and gene expression profile within 2 months. Moreover, PMT significantly improves alveolar protein deposition and other critical herPAP disease parameters. Thus, our data indicate iPSC-Mφ as a source of functional macrophages displaying substantial plasticity and therapeutic potential that upon pulmonary transplantation will integrate into the lung microenvironment, adopt an AMφ phenotype and gene expression pattern, and profoundly ameliorate pulmonary disease phenotypes.

Pulmonary Transplantation of Human Induced Pluripotent Stem Cell-derived Macrophages Ameliorates Pulmonary Alveolar Proteinosis.

Although the transplantation of induced pluripotent stem cell (iPSC)-derived cells harbors enormous potential for the treatment of pulmonary diseases, in vivo data demonstrating clear therapeutic benefits of human iPSC-derived cells in lung disease models are missing. We have tested the therapeutic potential of iPSC-derived macrophages in a humanized disease model of hereditary pulmonary alveolar proteinosis (PAP). Hereditary PAP is caused by a genetic defect of the GM-CSF (granulocyte-macrophage colony-stimulating factor) receptor, which leads to disturbed macrophage differentiation and protein/surfactant degradation in the lungs, subsequently resulting in severe respiratory insufficiency. Macrophages derived from human iPSCs underwent intrapulmonary transplantation into humanized PAP mice, and engraftment, in vivo differentiation, and therapeutic efficacy of the transplanted cells were analyzed. On intratracheal application, iPSC-derived macrophages engrafted in the lungs of humanized PAP mice. After 2 months, transplanted cells displayed the typical morphology, surface markers, functionality, and transcription profile of primary human alveolar macrophages. Alveolar proteinosis was significantly reduced as demonstrated by diminished protein content and surfactant protein D levels, decreased turbidity of the BAL fluid, and reduced surfactant deposition in the lungs of transplanted mice. We here demonstrate for the first time that pulmonary transplantation of human iPSC-derived macrophages leads to pulmonary engraftment, their in situ differentiation to an alveolar macrophage phenotype, and a reduction of alveolar proteinosis in a humanized PAP model. To our knowledge, this finding presents the first proof-of-concept for the therapeutic potential of human iPSC-derived cells in a pulmonary disease and may have profound implications beyond the rare disease of PAP.

TALEN-mediated functional correction of human iPSC-derived macrophages in context of hereditary pulmonary alveolar proteinosis

Hereditary pulmonary alveolar proteinosis (herPAP) constitutes a rare, life threatening lung disease characterized by the inability of alveolar macrophages to clear the alveolar airspaces from surfactant phospholipids. On a molecular level, the disorder is defined by a defect in the CSF2RA gene coding for the GM-CSF receptor alpha-chain (CD116). As therapeutic options are limited, we currently pursue a cell and gene therapy approach aiming for the intrapulmonary transplantation of gene-corrected macrophages derived from herPAP-specific induced pluripotent stem cells (herPAP-iPSC) employing transcriptional activator-like effector nucleases (TALENs). Targeted insertion of a codon-optimized CSF2RA-cDNA driven by the hybrid cytomegalovirus (CMV) early enhancer/chicken beta actin (CAG) promoter into the AAVS1 locus resulted in robust expression of the CSF2RA gene in gene-edited herPAP-iPSCs as well as thereof derived macrophages. These macrophages displayed typical morphology, surface phenotype, phagocytic and secretory activity, as well as functional CSF2RA expression verified by STAT5 phosphorylation and GM-CSF uptake studies. Thus, our study provides a proof-of-concept, that TALEN-mediated integration of the CSF2RA gene into the AAVS1 safe harbor locus in patient-specific iPSCs represents an efficient strategy to generate functionally corrected monocytes/macrophages, which in the future may serve as a source for an autologous cell-based gene therapy for the treatment of herPAP.

Function and Safety of Lentivirus-Mediated Gene Transfer for CSF2RA-Deficiency.

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder of pulmonary surfactant accumulation and hypoxemic respiratory failure caused by mutations in CSF2RA (encoding the granulocyte/macrophage colony-stimulating factor [GM-CSF] receptor α-chain [CD116]), which results in reduced GM-CSF-dependent pulmonary surfactant clearance by alveolar macrophages. While no pharmacologic therapy currently exists for hPAP, it was recently demonstrated that endotracheal instillation of wild-type or gene-corrected mononuclear phagocytes (pulmonary macrophage transplantation [PMT]) results in a significant and durable therapeutic efficacy in a validated murine model of hPAP. To facilitate the translation of PMT therapy to human hPAP patients, a self-inactivating (SIN) lentiviral vector was generated expressing a codon-optimized human CSF2RA-cDNA driven from an EF1α short promoter (Lv.EFS.CSF2RAcoop), and a series of nonclinical efficacy and safety studies were performed in cultured macrophage cell lines and primary human cells. Studies in cytokine-dependent Ba/F3 cells demonstrated efficient transduction, vector-derived CD116 expression proportional to vector copy number, and GM-CSF-dependent cell survival and proliferation. Using a novel cell line constructed to express a normal GM-CSF receptor β subunit and a dysfunctional α subunit (due to a function-altering CSF2RAG196R mutation) that reflects the macrophage disease phenotype of hPAP patients, it was demonstrated that Lv.EFS.CSF2RAcoop transduction restored GM-CSF receptor function. Further, Lv.EFS.CSF2RAcoop transduction of healthy primary CD34+ cells did not adversely affect cell proliferation or affect the cell differentiation program. Results demonstrate Lv.EFS.CSF2RAcoop reconstituted GM-CSF receptor α expression, restoring GM-CSF signaling in hPAP macrophages, and had no adverse effects in the intended target cells, thus supporting testing of PMT therapy of hPAP in humans.

Whole-Genome Sequencing of a Family with Hereditary Pulmonary Alveolar Proteinosis Identifies a Rare Structural Variant Involving CSF2RA/CRLF2/IL3RA Gene Disruption

Pulmonary alveolar proteinosis (PAP) is a rare pulmonary disease in which the abnormalities in alveolar surfactant accumulation are caused by impairments of GM-CSF pathway attributing to defects in a variety of genes. However, hereditary PAP is extremely uncommon and a detailed understanding in the genetic inheritance of PAP in a family may provide timely diagnosis, treatment and proper intervention including genetic consultation. Here, we described a comprehensive analysis of genome and gene expression for a family containing one affected child with a diagnosis of PAP and two other healthy siblings. Family-based whole-genome analysis revealed a homozygous deletion that disrupts CSF2RA, CRLF2, and IL3RA gene in the pseudoautosomal region of the X chromosome in the affected child and one of asymptomatic siblings. Further functional pathway analysis of differentially expressed genes in IL-1β-treated peripheral blood mononuclear cells highlighted the insufficiency of immune response in the child with PAP, especially the protection against bacterial infection. Collectively, our results reveal a novel allele as the genetic determinant of a family with PAP and provide insights into variable expressivity and incomplete penetrance of this rare disease, which will be helpful for proper genetic consultation and prompt treatment to avoid mortality and morbidity.

Elderly-onset hereditary pulmonary alveolar proteinosis and its cytokine profile

BackgroundPulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by surfactant accumulation, and is caused by disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. Abnormalities in CSF2 receptor alpha (CSF2RA) were reported to cause pediatric hereditary PAP. We report here the first case of CSF2RA-mutated, elderly-onset hereditary (h) PAP.Case presentationThe patient developed dyspnea on exertion, and was diagnosed with PAP at the age of 77 years, based on findings from chest computed tomography scan and bronchoalveolar lavage. She tested negative for GM-CSF autoantibodies, with no underlying disease. Her serum GM-CSF level was elevated (91.3 pg/mL), indicating GM-CSF signaling impairment and genetic defects in the GM-CSF receptor. GM-CSF-stimulated phosphorylation in signal transducer and activator of transcription 5 (STAT5) was not observed, and GM-CSF-Rα expression was defective in her blood cells. Genetic screening revealed a homozygous, single-base C > T mutation at nt 508—a nonsense mutation that yields a stop codon (Q170X)—in exon 7 of CSF2RA. High-resolution analysis of single nucleotide polymorphism array confirmed a 22.8-Mb loss of heterozygosity region in Xp22.33p22.11, encompassing the CSF2RA gene. She was successfully treated with whole lung lavage (WLL), which reduced the serum levels of interleukin (IL)-2, IL-5, and IL-17, although IL-3 and M-CSF levels remained high.ConclusionsThis is the first known report of elderly-onset hPAP associated with a CSF2RA mutation, which caused defective GM-CSF-Rα expression and impaired signaling. The analyses of serum cytokine levels during WLL suggested that GM-CSF signaling might be compensated by other signaling pathways, leading to elderly-onset PAP.

Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency.

SummaryInduced pluripotent stem cells (iPSCs) represent an innovative source for the standardized in vitro generation of macrophages (Mφ). We here describe a robust and efficient protocol to obtain mature and functional Mφ from healthy as well as disease-specific murine iPSCs. With regard to morphology, surface phenotype, and function, our iPSC-derived Mφ (iPSC-Mφ) closely resemble their counterparts generated in vitro from bone marrow cells. Moreover, when we investigated the feasibility of our differentiation system to serve as a model for rare congenital diseases associated with Mφ malfunction, we were able to faithfully recapitulate the pathognomonic defects in GM-CSF signaling and Mφ function present in hereditary pulmonary alveolar proteinosis (herPAP). Thus, our studies may help to overcome the limitations placed on research into certain rare disease entities by the lack of an adequate supply of disease-specific primary cells, and may aid the development of novel therapeutic approaches for herPAP patients.

35. Establishment of the Dose-Response Relationship Needed for Human Translation of Pulmonary Macrophage Transplantation (PMT) Therapy of Hereditary Pulmonary Alveolar Proteinosis (hPAP)

Hereditary pulmonary alveolar proteinosis (hPAP) is a severe pediatric lung disease caused by mutations in CSF2RA/B (encoding GM-CSF receptor α/β, respectively) without pharmacologic therapy. Disease patho-genesis is mediated by loss of GM-CSF-dependent clearance by alveolar macrophages (AMs) resulting in progressive pulmonary alveolar surfactant accumulation and hypoxemic respiratory failure. We recently reported a conceptually and technically novel, exceptionally efficient gene and cell therapy approach -PMT- as a promising alternative for the existing highly invasive, inefficient whole lung lavage procedure for hPAP. Our preclinical studies revealed that macrophage cell doses from 0.5 to 4 x106/mouse were similarly highly efficacious as therapy of hPAP in Csf2rb-/- mice, an authentic model of human hPAP (Nature, 2014, 514: 450-5). The present study was undertaken to identify a dose-response relationship and to address the hypothesis of an expected ‘trade-off’ between the minimum effective dose and time to treatment effect. We isolated Lineage-, Sca1+, cKit+ hematopoietic stem/progenitors from the bone marrow of wild-type (WT) CD45.1+ mice, differentiated them in vitro into mature macrophages and sorted for highly homogenous CD11cHi, F4/80Hi macrophage population by flow cytometry. We administered cell doses (2.5 x103, 2.5 x104, 2.5 x105, 1 x106 cells/mouse; n=3-4/dose) by PMT to CD45.2+ Csf2rb-/- recipients. At 8 weeks after PMT, therapeutic efficacy was evaluated by measuring the optical density (OD) of bronchoalveolar lavage (BAL turbidity) - an excellent measure of overall PAP disease severity. The CD45.1+ donor macrophages recovered from the BAL were of CD11cHi, F4/80Hi phenotype. BAL turbidity decreased smoothly with increasing PMT cell dose over the entire range but reached significance only at the two highest doses compared to age-matched, untreated Csf2rb-/- controls (OD λ=600 nm = 0.88±0.07 and 0.5±0.08 vs. 2.32±0.18; P<0.001, P<0.0001; respectively). BAL turbidity correlated inversely with the percentage of CD45.1+ donor macrophage engraftment in CD45.2+ Csf2rb-/- recipients (Spearman rank correlation, R2=0.74). Since we have identified an increased ratio of cholesterol (relative to phospholipid) in surfactant as the primary lipid abnormality in hPAP, we also evaluated the relationship of BAL cholesterol level and PMT cell dose. Like turbidity, BAL cholesterol levels decreased smoothly with PMT cell dose over the entire range but reached significance only at the two highest cell doses compared to age-matched, untreated Csf2rb-/- controls (ng/ml BAL = 25±3 and 10±0.3 versus 54±0.3; P<0.01, P<0.001; respectively, R2=0.67). Our pre-clinical results establish a direct relationship between the number of macrophages transplanted and efficacy of PMT therapy in Csf2rb-/- mice and facilitate the design of a clinical trial to test PMT therapy in children with hPAP. Based on these results and the previously demonstrated strong survival advantage of transplanted over endogenous macrophages, we anticipate a ‘trade-off’ between the minimum effective dose and the time to equivalent treatment effect.

522. Murine iPSC-Derived Macrophages Improve the In Vivo Disease Phenotype of Pulmonary Alveolar Proteinosis Due to Csf2rb Deficiency

Induced pluripotent stem cells (iPSCs) have proven applicability to various areas including disease modeling and cell therapy. Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations within the α- (CSF2RA) or β-chain (CSF2RB) of the GM-CSF receptor gene (CSF2R), resulting in the inability of alveolar macrophages (MΦ) to clear the alveolar spaces from surfactant material. Given the limited treatment options in herPAP, we evaluated the suitability of murine iPSC-derived MΦ (iPSC-MΦ) for disease modelling and as a source for an innovative cell replacement therapy. To this point we first established an efficient and robust protocol to obtain mature and functional MΦ from healthy murine iPSCs. These MΦ closely resemble their counterparts generated in vitro from bone marrow cells with regard to morphology, surface phenotype and function. Furthermore, murine Csf2rb-deficient iPSCs (miPAP) were generated and submitted to our differentiation protocol. miPAP-derived MΦ faithfully recapitulated the defects in GM-CSF signaling and MΦ function present in herPAP patients. Of note, we also evaluated the feasibility, safety and clinical benefit of the intratracheal application of healthy iPSC-MΦ using a clinically relevant in vivo mouse model of herPAP. Following single pulmonary MΦ transplantation (PMT) of 4×106 iPSC-MΦ, specific engraftment was observed in the alveolar spaces for up to 8 weeks as shown by tissue sections and PCR. Moreover, cells displayed donor-specific CD45.1 expression and typical MΦ morphology in vivo and upon re-isolation. No teratoma formation or tissue toxicity was detected in the organs of transplanted mice. Most importantly, following PMT a significant improvement of disease parameters such as reduced protein, M-CSF, GM-CSF and surfactant protein-D (SP-D) concentration was shown in the bronchoalveolar lavage fluid. We also observed a decrease in PAS-positive material in lung sections and a reduction in lung opacity in computer tomography scans. Thus, we here present an efficient differentiation protocol to obtain MΦ from iPSCs for disease modeling and introduce PMT of iPSC-MΦ as an innovative cell therapy for herPAP.