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Primary or secondary immunodeficiencies are characterized by disruption of cellular and humoral immunity. Respiratory infections are a major cause of morbidity and mortality among immunodeficient or immunocompromised patients, with Staphylococcus aureus being a common offending organism. We propose here an adoptive macrophage transfer approach aiming to enhance impaired pulmonary immunity against S aureus. Our studies, using human-induced pluripotent stem cell-derived macrophages (iMφs), demonstrate efficient antimicrobial potential against methicillin-sensitive and methicillin-resistant clinical isolates of S aureus. Using an S aureus airway infection model in immunodeficient mice, we demonstrate that the adoptive transfer of iMφs is able to reduce the bacterial load more than 10-fold within 20 hours. This effect was associated with reduced granulocyte infiltration and less damage in lung tissue of transplanted animals. Whole transcriptome analysis of iMφs compared with monocyte-derived macrophages indicates a more profound upregulation of inflammatory genes early after infection and faster normalization 24 hours postinfection. Our data demonstrate high therapeutic efficacy of iMφ-based immunotherapy against S aureus infections and offer an alternative treatment strategy for immunodeficient or immunocompromised patients.

Inherited deficiency of the antiprotease alpha-1 antitrypsin (AAT) is associated with liver failure and early-onset emphysema. In mice, in vivo lentiviral transduction of alveolar macrophages (AMs) has been described to yield protective pulmonary AAT levels and ameliorate emphysema development. We here investigated the pulmonary transplantation of macrophages (PMT) transgenic for AAT as a potential therapy for AAT deficiency-associated lung pathology. Employing third-generation SIN-lentiviral vectors expressing the human AAT cDNA from the CAG or Cbx-EF1α promoter, we obtained high-level AAT secretion in a murine AM cell line as well as murine bone marrow-derived macrophages differentiated in vitro (AAT MΦ). Secreted AAT demonstrated a physiologic glycosylation pattern as well as elastase-inhibitory and anti-apoptotic properties. AAT MΦ preserved normal morphology, surface phenotype, and functionality. Furthermore, in vitro generated murine AAT MΦ successfully engrafted in AM-deficient Csf2rb–/–mice and converted into a CD11c+/Siglec-F+AM phenotype as detected in bronchoalveolar lavage fluid and homogenized lung tissue 2 months after PMT. Moreover, human AAT was detected in the lung epithelial lining fluid of transplanted animals. Efficient AAT expression and secretion were also demonstrated for human AAT MΦ, confirming the applicability of our vectors in human cells.

Autosomal recessive (AR) complete interferon-γ receptor 1 (IFN-γR1) deficiency, also known as one genetic etiology of Mendelian susceptibility to mycobacterial disease (MSMD), is a life-threatening congenital disease leading to premature death. Affected patients present a pathognomonic predisposition to recurrent and severe infections with environmental mycobacteria or the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine. Current therapeutic options are limited to antibiotic treatment and hematopoietic stem cell transplantation, however with poor outcome. Given the clinical success of gene therapy, we introduce the first lentiviral-based gene therapy approach to restore expression and function of the human IFN-γR-downstream signaling cascade. In our study, we developed lentiviral vectors constitutively expressing the human IFN-γR1 and demonstrate stable transgene expression without interference with cell viability and proliferation in transduced human hematopoietic cells. Using an IFN-γR1-deficient HeLa cell model, we show stable receptor reconstitution and restored IFN-γR1 signaling without adverse effect on cell functionality. Transduction of both SV40-immortalized and primary fibroblasts derived from IFN-γR1-deficient MSMD patients was able to recover IFN-γR1 expression and restore type II IFN signaling upon stimulation with IFN-γ. In summary, we highlight lentiviral vectors to correct the IFN-γ mediated immunity and present the first gene therapy approach for patients suffering from AR complete IFN-γR1 deficiency., Graphical Abstract, Current therapeutic options for IFNγR1-deficient Mendelian susceptibility to mycobacterial disease (MSMD) are limited to allogenic bone marrow transplantation. This study highlights lentiviral vectors to correct the cellular phenotype of IFNγR1-deficient HeLa cells and primary patient cells, paving the way for gene therapy strategies.

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.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to impaired ion transport in epithelial cells. Although lung failure due to chronic infection is the major comorbidity in individuals with cystic fibrosis, the role of CFTR in non-epithelial cells has not been definitively resolved. Given the important role of host defense cells, we evaluated the Cftr deficiency in pulmonary immune cells by hematopoietic stem cell transplantation in cystic fibrosis mice. We transplanted healthy bone marrow stem cells and could reveal a stable chimerism of wild-type cells in peripheral blood. The outcome of stem cell transplantation and the impact of healthy immune cells were evaluated in acute Pseudomonas aeruginosa airway infection. In this study, mice transplanted with wild-type cells displayed better survival, lower lung bacterial numbers, and a milder disease course. This improved physiology of infected mice correlated with successful intrapulmonary engraftment of graft-derived alveolar macrophages, as seen by immunofluorescence microscopy and flow cytometry of graft-specific leucocyte surface marker CD45 and macrophage marker CD68. Given the beneficial effect of hematopoietic stem cell transplantation and stable engraftment of monocyte-derived CD68-positive macrophages, we conclude that replacement of mutant Cftr macrophages attenuates airway infection in cystic fibrosis mice.

Induced pluripotent stem cells (iPSCs) offer great promise for the field of regenerative medicine, and iPSC-derived cells have already been applied in clinical practice. However, potential contamination of effector cells with residual pluripotent cells (e.g., teratoma-initiating cells) or effector cell-associated side effects may limit this approach. This also holds true for iPSC-derived hematopoietic cells. Given the therapeutic benefit of macrophages in different disease entities and the feasibility to derive macrophages from human iPSCs, we established human iPSCs harboring the inducible Caspase-9 (iCasp9) suicide safety switch utilizing transcription activator-like effector nuclease (TALEN)-based designer nuclease technology. Mono- or bi-allelic integration of the iCasp9 gene cassette into the AAVS1 locus showed no effect on the pluripotency of human iPSCs and did not interfere with their differentiation towards macrophages. In both, iCasp9-mono and iCasp9-bi-allelic clones, concentrations of 0.1 nM AP20187 were sufficient to induce apoptosis in more than 98% of iPSCs and their progeny&mdash, macrophages. Thus, here we provide evidence that the introduction of the iCasp9 suicide gene into the AAVS1 locus enables the effective clearance of human iPSCs and thereof derived macrophages.

Summary: Induced 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. : Mucci and colleagues demonstrate marked plasticity of iPSC-derived macrophages and rapid adoption of an alveolar macrophage phenotype upon pulmonary transplantation, as well as profound therapeutic efficacy of iPSC-derived macrophages in the context of the severe lung disease pulmonary alveolar proteinosis. Key words: iPSC, hematopoiesis, macrophages, lung, cell therapy

Mendelian susceptibility to mycobacterial disease is a rare primary immunodeficiency characterized by severe infections caused by weakly virulent mycobacteria. Biallelic null mutations in genes encoding interferon gamma receptor 1 or 2 (IFNGR1 or IFNGR2) result in a life-threatening disease phenotype in early childhood. Recombinant interferon γ (IFN-γ) therapy is inefficient, and hematopoietic stem cell transplantation has a poor prognosis. Thus, we developed a hematopoietic stem cell (HSC) gene therapy approach using lentiviral vectors that express Ifnγr1 either constitutively or myeloid specifically. Transduction of mouse Ifnγr1-/- HSCs led to stable IFNγR1 expression on macrophages, which rescued their cellular responses to IFN-γ. As a consequence, genetically corrected HSC-derived macrophages were able to suppress T-cell activation and showed restored antimycobacterial activity against Mycobacterium avium and Mycobacterium bovis Bacille Calmette-Guerin (BCG) in vitro. Transplantation of genetically corrected HSCs into Ifnγr1-/- mice before BCG infection prevented manifestations of severe BCG disease and maintained lung and spleen organ integrity, which was accompanied by a reduced mycobacterial burden in lung and spleen and a prolonged overall survival in animals that received a transplant. In summary, we demonstrate an HSC-based gene therapy approach for IFNγR1 deficiency, which protects mice from severe mycobacterial infections, thereby laying the foundation for a new therapeutic intervention in corresponding human patients.

The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45+CD11b+CD14+CD163+ cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa-mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies., Pulmonary infections constitute a substantial health problem worldwide. Here the authors show that phagocytes similar to primitive macrophages can be generated from human induced pluripotent stem cells, by the use of industry-compatible, stirred-tank bioreactors, and applied as a cell-based therapy to treat acute bacterial infections in mice.

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.

Hereditary pulmonary alveolar proteinosis (hPAP) caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (CSF2RA) deficiency is a rare, life-threatening lung disease characterized by accumulation of proteins and phospholipids in the alveolar spaces. The disease is caused by a functional insufficiency of alveolar macrophages, which require GM-CSF signaling for terminal differentiation and effective degradation of alveolar proteins and phospholipids. Therapeutic options are extremely limited, and the pathophysiology underlying the defective protein degradation in hPAP alveolar macrophages remains poorly understood.To further elucidate the cellular mechanisms underlying hPAP and evaluate novel therapeutic strategies, we here investigated the potential of hPAP patient-derived induced pluripotent stem cell (PAP-iPSCs) derived monocytes and macrophages.Patient-specific PAP-iPSCs were generated from CD34(+) bone marrow cells of a CSF2RA-deficient patient with PAP. We assessed pluripotency, chromosomal integrity, and genetic correction of established iPSC lines. On hematopoietic differentiation, genetically corrected or noncorrected monocytes and macrophages were investigated in GM-CSF-dependent assays.Although monocytes and macrophages differentiated from noncorrected PAP-iPSCs exhibited distinct defects in GM-CSF-dependent functions, such as perturbed CD11b activation, phagocytic activity, and STAT5 phosphorylation after GM-CSF exposure and lack of GM-CSF uptake, these defects were fully repaired on lentiviral gene transfer of a codon-optimized CSF2RA-cDNA.These data establish PAP-iPSC-derived monocytes and macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes and macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to patients with hPAP could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.

Background Hematologic toxicity represents a major side effect of cytotoxic chemotherapy frequently preventing adequately dosed chemotherapy application and impeding therapeutic success. Transgenic (over)expression of chemotherapy resistance (CTX-R) genes in hematopoietic stem- and progenitor cells represents a potential strategy to overcome this problem. To apply this concept in the context of acute myeloid leukemia and myelodysplasia, we have investigated the overexpression of the multidrug resistance 1 (MDR1) and the cytidine deaminase (CDD) gene conferring resistance to anthracyclines and cytarabine (Ara-C), the two most important drugs in the treatment of these diseases. Methods State-of-the-art, third generation, self-inactivating (SIN) lentiviral vectors were utilized to overexpress a human CDD-cDNA and a codon-optimized human MDR1-cDNA corrected for cryptic splice sites from a spleen focus forming virus derived internal promoter. Studies were performed in myeloid 32D cells as well as primary lineage marker negative (lin−) murine bone marrow cells and flow cytometric analysis of suspension cultures and clonogenic analysis of vector transduced cells following cytotoxic drug challenge were utilized as read outs. Results Efficient chemoprotection of CDD and MDR1 transduced hematopoietic 32D as well as primary lin− cells was proven in the context of Ara-C and anthracycline application. Both, CTX-R transduced 32D as well as primary hematopoietic cells displayed marked resistance at concentrations 5–20 times the LD50 of non-transduced control cells. Moreover, simultaneous CDD/MDR1 gene transfer resulted in similar protection levels even when combined Ara-C anthracycline treatment was applied. Furthermore, significant enrichment of transduced cells was observed upon cytotoxic drug administration. Conclusions Our data demonstrate efficient chemoprotection as well as enrichment of transduced cells in hematopoietic cell lines as well as primary murine hematopoietic progenitor cells following Ara-C and/or anthracycline application, arguing for the efficacy as well as feasibility of our approach and warranting further evaluation of this concept. Electronic supplementary material The online version of this article (doi:10.1186/s13046-015-0260-4) contains supplementary material, which is available to authorized users.

Contains fulltext : 138569.pdf (Publisher’s version ) (Open Access) The mitochondrial kinase PINK1 and the ubiquitin ligase Parkin are participating in quality control after CCCP- or ROS-induced mitochondrial damage, and their dysfunction is associated with the development and progression of Parkinson's disease. Furthermore, PINK1 expression is also induced by starvation indicating an additional role for PINK1 in stress response. Therefore, the effects of PINK1 deficiency on the autophago-lysosomal pathway during stress were investigated. Under trophic deprivation SH-SY5Y cells with stable PINK1 knockdown showed downregulation of key autophagic genes, including Beclin, LC3 and LAMP-2. In good agreement, protein levels of LC3-II and LAMP-2 but not of LAMP-1 were reduced in different cell model systems with PINK1 knockdown or knockout after addition of different stressors. This downregulation of autophagic factors caused increased apoptosis, which could be rescued by overexpression of LC3 or PINK1. Taken together, the PINK1-mediated reduction of autophagic key factors during stress resulted in increased cell death, thus defining an additional pathway that could contribute to the progression of Parkinson's disease in patients with PINK1 mutations.

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.

Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a rare primary immunodeficiency characterized by recurrent severe infections with otherwise only weakly virulent mycobacteria. MSMD is associated with mutations in different genes, all leading to an impaired activation of macrophages by T-cells and a defective innate immune response. Specifically mutations in the interferon-gamma (IFNγ)-receptor-1 or-2 (IFNγR1 /2) genes result in a life-threatening disease phenotype with most patients dying in early childhood. We here introduce a novel gene therapy approach for IFNγR1 deficiency. Thus, we have designed a 3rd generation SIN lentiviral vector expressing the murine cDNA of Ifnγr1 from a spleen focus forming virus (SFFV) promoter element coupled by an IRES to GFP (Lv.SFFV.Ifnyr1.iGFP). Transduction of hematopoietic stem/progenitor cells from Ifnγrl−/− mice showed expression of Ifnγr1 by FACS and qRT-PCR and no abnormalities in clonogenic growth when compared to WT control cells. Moreover, differentiation of transduced cells towards macrophages (MΦ) by M-CSF was normal as determined by morphology on cytospins and surface marker expression of CD11b, CD200R, CD115, CD45 and F4/80. When subjecting corrected MΦ to stimulation with IFNγ, corrected cells were able to build a functional IFNγR1-Ifnγ-IFNγR2 and internalize IFNγ as efficiently as WT cells, as suggested by the clearance of IFNγ from the medium within 24h of stimulation. Consequently, Lv. SFFV. Ifnγr1.iGFP corrected MΦ revealed restored up-regulation of HLA-DR and CD86 (B7.2) comparable to WT-MΦ. IFNγ dependent T-cell activation was evaluated using T cell receptor- transgenic T cells recognizing ovalbumin (OVA). Both WT and corrected MΦ were unable to activate T-cells in the presence of IFNγ and OVA, whereas MΦ from Ifnγr1−/− mice induced significantly stronger proliferation of T cells. This observation was accompanied by the induction of indoleamine 2,3-dioxygenase (IDO) in WT and corrected MΦ, suggesting that IDO enables MΦ to deplete tryptophan from the medium and interferes with T-cell proliferation. Finally, Ifnγrl downstream signaling showed restored phosphorylation of STAT1 in corrected MΦ, consistent with induction of iNos and Irf1 upon stimulation with IFNγ. Moreover, correction of Ifnγrl−/− MΦ led to a significantly improved anti-mycobacterial activity as measured by comparable killing of Mycobacterium Avium in corrected and WT MΦ. Thus, we here introduce a novel gene therapy approach for MSMD in the context of Ifnγr1 deficiency.

Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte/macrophage-colony-stimulating factor (GM-CSF) receptor genes (CSF2RA and CSF2RB), resulting in disturbed alveolar macrophage (AM) differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. We here introduce pulmonary transplantation of gene corrected hematopoietic stem cell (HSC)-derived macrophage progenitors (MP) as a novel, cause directed, and well-tolerated therapy for herPAP. In a Csf2rb-/- mouse-model, selective pulmonary engraftment of healthy donor cells upon pulmonary transplantation of MPs was demonstrated by flow- and chip cytometry. Profound reduction of alveolar-protein levels and significant improvement of clinical parameters such as lung function and lung densities on CT scans were observed for more than nine months. Subsequent in situ analysis of donor cells revealed in vivo differentiation towards an AM phenotype characterized by CD11chi, CD11blo, MHC-II+, CD14+, F4/80+ surface marker, poor antigen presentation capacity, high phagocytic activity and AM-typical morphology on electron microscopy. Similar results were obtained following pulmonary transplantation of MPs differentiated from lentivirally corrected Csf2rb -/- HSCs. In vitro these gene-corrected HSCs expanded up to 1045-fold while differentiating into typical alveolar macrophages with F4/80, CD11b, CD11c, CD68, as well as Csf2rb mRNA and protein (CD131) expression and reconstitution of GM-CSF receptor signaling. Transplanted herPAP mice displayed significant improvement of biomarkers in the bronchoalveolar fluid (cloudiness, turbidity, SP-D, MCP-1, M-CSF, and GM-CSF) and in AMs (mRNA for PU.1, PPARg and ABCG1). Moreover, administration of human CD34+ cell-derived MPs profoundly improved symptoms in a humanized herPAP mouse model. Here, transplantation of 1-2x106 human MPs led to long-term pulmonary engraftment and reduced alveolar protein levels by 50-70%. CT scans six months after transplantation revealed significant improvements in herPAP related signs, including marked reduction of expiratory lung densities and normalisation of inspiratory to expiratory lung volume ratio. Furthermore, to genetically correct human CSF2RA-patient derived CD34+ cells, we have generated SIN-lentiviral vectors expressing a codon-optimized human CSF2RA-cDNA in combination with EGFP (Lv.EFS.CSF2RA.EGFP) from an EFS1a-promoter. BaF3 cells transduced with this vector showed stable and longterm (>3 month) expression of CSF2RA (CD116) by flow cytometry and survived in hGM-CSF dependant assays even at low concentrations of GM-CSF (5 ng/ml) confirming the formation of functional hybrid receptors of the murine GM-CSF receptor ß-chain with the transgene. Characterization of GM-CSF receptor downstream signalling revealed 5-6-fold increased STAT5 phosphorylation by Western blot in response to hGM-CSF (over control cells). Conferring this vector to CD34+ cells of CSF2RA-deficient patients yielded efficient CD116 (CSF2RA) expression, and rescued hGM-CSF dependent colony formation as well as monocytoid differentiation. Of note, clonogenic growth by G-CSF control treatment revealed no differences in colony formation or differentiation capacity when compared to uncorrected patient samples. Furthermore, healthy Lv.EFS.CSF2RA.EGFP transduced CD34+ samples, while showing robust CD116 overexpression, exhibited no aberrations in biological functions such as colony formation or in vitro differentiation towards macrophages. Thus, we here describe an innovative, cause directed and highly effective hematopoietic gene therapy approach to herPAP. Given the longevity of the transplanted MP population in our model, the strategy also may serve as a proof-of-principle to incorporate long-lived differentiated, cell sources into current hematopoietic gene therapy concepts. Disclosures No relevant conflicts of interest to declare.