Paritha Arumugam

Pulmonary macrophage transplantation therapy

Bone-marrow transplantation is an effective cell therapy but requires myeloablation, which increases infection risk and mortality. Recent lineage-tracing studies documenting that resident macrophage populations self-maintain independently of haematological progenitors prompted us to consider organ-targeted, cell-specific therapy. Here, using granulocyte–macrophage colony-stimulating factor (GM-CSF) receptor-β-deficient (Csf2rb−/−) mice that develop a myeloid cell disorder identical to hereditary pulmonary alveolar proteinosis (hPAP) in children with CSF2RA or CSF2RB mutations, we show that pulmonary macrophage transplantation (PMT) of either wild-type or Csf2rb-gene-corrected macrophages without myeloablation was safe and well-tolerated and that one administration corrected the lung disease, secondary systemic manifestations and normalized disease-related biomarkers, and prevented disease-specific mortality. PMT-derived alveolar macrophages persisted for at least one year as did therapeutic effects. Our findings identify mechanisms regulating alveolar macrophage population size in health and disease, indicate that GM-CSF is required for phenotypic determination of alveolar macrophages, and support translation of PMT as the first specific therapy for children with hPAP.

Genotoxic Potential of Lineage-specific Lentivirus Vectors Carrying the β-Globin Locus Control Region

Insertional mutagenesis by long terminal repeat (LTR) enhancers in γ-retrovirus-based vectors (GVs) in clinical trials has prompted deeper investigations into vector genotoxicity. Experimentally, self-inactivating (SIN) lentivirus vectors (LVs) and GV containing internal promoters/enhancers show reduced genotoxicity, although strong ubiquitously-active enhancers dysregulate genes independent of vector type/design. Herein, we explored the genotoxicity of β-globin (BG) locus control region (LCR), a strong long-range lineage-specific-enhancer, with/without insulator (Ins) elements in LV using primary hematopoietic progenitors to generate in vitro immortalization (IVIM) assay mutants. LCR-containing LV had ~200-fold lower transforming potential, compared to the conventional GV. The LCR perturbed expression of few genes in a 300 kilobase (kb) proviral vicinity but no upregulation of genes associated with cancer, including an erythroid-specific transcription factor occurred. A further twofold reduction in transforming activity was observed with insulated LCR-containing LV. Our data indicate that toxicology studies of LCR-containing LV in mice will likely not yield any insertional oncogenesis with the numbers of animals that can be practically studied.Insertional mutagenesis by long terminal repeat (LTR) enhancers in gamma-retrovirus-based vectors (GVs) in clinical trials has prompted deeper investigations into vector genotoxicity. Experimentally, self-inactivating (SIN) lentivirus vectors (LVs) and GV containing internal promoters/enhancers show reduced genotoxicity, although strong ubiquitously-active enhancers dysregulate genes independent of vector type/design. Herein, we explored the genotoxicity of beta-globin (BG) locus control region (LCR), a strong long-range lineage-specific-enhancer, with/without insulator (Ins) elements in LV using primary hematopoietic progenitors to generate in vitro immortalization (IVIM) assay mutants. LCR-containing LV had approximately 200-fold lower transforming potential, compared to the conventional GV. The LCR perturbed expression of few genes in a 300 kilobase (kb) proviral vicinity but no upregulation of genes associated with cancer, including an erythroid-specific transcription factor occurred. A further twofold reduction in transforming activity was observed with insulated LCR-containing LV. Our data indicate that toxicology studies of LCR-containing LV in mice will likely not yield any insertional oncogenesis with the numbers of animals that can be practically studied.

The 3′ Region of the Chicken Hypersensitive Site-4 Insulator Has Properties Similar to Its Core and Is Required for Full Insulator Activity

Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the “core”, which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5′ core with a novel 3′ 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors.

Perforin Gene Transfer Into Hematopoietic Stem Cells Improves Immune Dysregulation in Murine Models of Perforin Deficiency

Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8+ T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8+ lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL.

Placenta growth factor augments airway hyperresponsiveness via leukotrienes and IL-13

Airway hyperresponsiveness (AHR) affects 55%-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the mechanisms underlying the high AHR prevalence in a hemoglobinopathy remain unknown. We hypothesized that placenta growth factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates AHR. In allergen-exposed mice, loss of Plgf dampened AHR, reduced inflammation and eosinophilia, and decreased expression of the Th2 cytokine IL-13 and the leukotriene-synthesizing enzymes 5-lipoxygenase and leukotriene-C4-synthase. Plgf-/- mice treated with leukotrienes phenocopied the WT response to allergen exposure; conversely, anti-PlGF Ab administration in WT animals blunted the AHR. Notably, Th2-mediated STAT6 activation further increased PlGF expression from lung epithelium, eosinophils, and macrophages, creating a PlGF/leukotriene/Th2-response positive feedback loop. Similarly, we found that the Th2 response in asthma patients is associated with increased expression of PlGF and its downstream genes in respiratory epithelial cells. In an SCD mouse model, we observed increased AHR and higher leukotriene levels that were abrogated by anti-PlGF Ab or the 5-lipoxygenase inhibitor zileuton. Overall, our findings indicate that PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. These data also suggest that zileuton and anti-PlGF Ab could be promising therapies to reduce pulmonary morbidity in SCD.

Gene Therapy for β-Thalassemia

Gene transfer for beta-thalassemia requires gene transfer into hematopoietic stem cells using integrating vectors that direct regulated expression of beta globin at therapeutic levels. Among integrating vectors, oncoretroviral vectors carrying the human beta-globin gene and portions of the locus control region (LCR) have suffered from problems of vector instability, low titers and variable expression. In recent studies, human immunodeficiency virus-based lentiviral (LV) vectors were shown to stably transmit the human beta-globin gene and a large LCR element, resulting in correction of beta-thalassemia intermedia in mice. Several groups have since demonstrated correction of the mouse thalassemia intermedia phenotype, with variable levels of beta-globin expression. These levels of expression were insufficient to fully correct the anemia in thalassemia major mouse model. Insertion of a chicken hypersensitive site-4 chicken insulator element (cHS4) in self-inactivating (SIN) LV vectors resulted in higher and less variable expression of human beta-globin, similar to the observations with cHS4-containing retroviral vectors carrying the human gamma-globin gene. The levels of beta-globin expression achieved from insulated SIN-LV vectors were sufficient to phenotypically correct the thalassemia phenotype from 4 patients with human thalassemia major in vitro, and this correction persisted long term for up to 4 months, in xeno-transplanted mice in vivo. In summary, LV vectors have paved the way for clinical gene therapy trials for Cooleys anemia and other beta-globin disorders. SIN-LV vectors address several safety concerns of randomly integrating viral vectors by removing viral transcriptional elements and providing lineage-restricted expression. Flanking the proviral cassette with chromatin insulator elements, which additionally have enhancer-blocking properties, may further improve SIN-LV vector safety.

Pigtailed macaques as a model to study long-term safety of lentivirus vector-mediated gene therapy for hemoglobinopathies

Safely achieving long-term engraftment of genetically modified hematopoietic stem cells (HSCs) that maintain therapeutic transgene expression is the benchmark for successful application of gene therapy for hemoglobinopathies. We used the pigtailed macaque HSC transplantation model to ascertain the long-term safety and stability of a γ-globin lentivirus vector. We observed stable gene-modified cells and fetal hemoglobin expression for 3 years. Retrovirus integration site (RIS) analysis spanning 6 months to 3.1 years revealed vastly disparate integration profiles, and dynamic fluctuation of hematopoietic contribution from different gene-modified HSC clones without evidence for clonal dominance. There were no perturbations of the global gene-expression profile or expression of genes within a 300 kb region of RIS, including genes surrounding the most abundantly marked clones. Overall, a 3-year long follow-up revealed no evidence of genotoxicity of the γ-globin lentivirus vector with multilineage polyclonal hematopoiesis, and HSC clonal fluctuations that were not associated with transcriptome dysregulation.

Targeting cholesterol homeostasis in lung diseases

Macrophages are critical to organ structure and function in health and disease. To determine mechanisms by which granulocyte/macrophage-colony stimulating factor (GM-CSF) signaling normally maintains surfactant homeostasis and how its disruption causes pulmonary alveolar proteinosis (PAP), we evaluated lipid composition in alveolar macrophages and lung surfactant, macrophage-mediated surfactant clearance kinetics/dynamics, and cholesterol-targeted pharmacotherapy of PAP in vitro and in vivo. Without GM-CSF signaling, surfactant-exposed macrophages massively accumulated cholesterol ester-rich lipid-droplets and surfactant had an increased proportion of cholesterol. GM-CSF regulated cholesterol clearance in macrophages in constitutive, dose-dependent, and reversible fashion but did not affect phospholipid clearance. PPARγ-agonist therapy increased cholesterol clearance in macrophages and reduced disease severity in PAP mice. Results demonstrate that GM-CSF is required for cholesterol clearance in macrophages, identify reduced cholesterol clearance as the primary macrophage defect driving PAP pathogenesis, and support the feasibility of translating pioglitazone as a novel pharmacotherapy of PAP.

21. Pulmonary Macrophage Transplantation (PMT) Therapy of Hereditary Pulmonary Alveolar Proteinosis (hPAP) Is Effective with Mature Macrophages and Does Not Require Myeloid Precursor/Progenitor Expansion

Rationale: Hereditary Pulmonary Alveolar Proteinosis (hPAP) is caused by mutations in the CSF2RA or CSF2RB genes (encoding GM-CSF receptor α or β, respectively) leading to defective GM-CSF-dependent surfactant clearance by alveolar macrophages, resulting in severe respiratory failure. Recently, using a validated model of hPAP (Csf2rb−/− mice), we reported a novel Pulmonary Macrophage Transplantation (PMT) approach as a safe, effective, tissue-specific and durable therapy for hPAP disease (Nature. 2014, 514: 450). Our results showed that wild-type bone marrow-derived macrophages (BMDMs) delivered by PMT successfully engraft, proliferate, gradually replace functionally deficient endogenous alveolar macrophages and efficiently clear surfactant in Csf2rb−/−recipient mice resulting in durable hPAP disease correction, in the absence of myeloablation. However, these studies did not exclude the possibility of an expanded myeloid progenitor population as the effector cell in PMT therapy of hPAP. Because the transplanted cells were capable of clearing surfactant, we hypothesized that the therapeutic efficacy of PMT is mediated by mature macrophages without obligate expansion of any myeloid progenitor/stem cell. Methods: BMDMs or alveolar macrophages were obtained from wild-type mice and intrapulmonary administered into Csf2rb−/− mice by PMT (2.5×105 per mouse). The therapeutic efficacy was evaluated at 2 months after PMT by measuring the optical density (OD λ=600 nm) of bronchoalveolar lavage (BAL) turbidity - an excellent measure of overall PAP disease severity. Results: Characterization prior to transplantation showed that BMDMs were highly purified, mature macrophages: they had the morphologic appearance and surface markers of mature macrophages, clonogenic analysis indicated they contained less than 0.005% CFU-GM and no BFU-E, or CFU-GEMM progenitors, and they were able to clear surfactant in vitro. To further increase macrophage purity, BMDMs were sorted by flow cytometer by applying a conservative gating strategy to isolate CD11cHiF4/80Hi macrophages. PMT using these highly uniform, mature CD11cHiF4/80Hi BMDMs into Csf2rb−/− mice showed extraordinary therapeutic efficacy as evidenced by marked reduction of BAL turbidity compared to untreated, age-matched Csf2rb−/− mice (OD = 0.9±0.1 vs 2.3±0.2; n=4; P<0.01). As a second approach to exclude the possibility of an expanded hematopoietic progenitor as the therapeutic effector cell of PMT therapy, highly purified CD11cHiF4/80Hi alveolar macrophages were delivered by PMT into Csf2rb−/− mice. Mature alveolar macrophages were equally efficacious in hPAP disease correction, as shown by the marked reduction of BAL turbidity compared to untreated, age-matched Csf2rb−/− mice (OD = 1.5±0.1 vs 2.3±0.2; n=4; P<0.01). All donor cells analyzed at 2 months after PMT were of CD11cHiF4/80Hi phenotype. Conclusions: Results demonstrate that mature macrophages are the effector cells of PMT therapy, excluding a requirement for progenitor population expansion in hPAP disease correction. These observations have important implications in clinical trial design for translating PMT therapy to human children with hPAP.

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.