Elimination of the fibrinogen integrin αMß2-binding motif improves renal pathology in mice with sickle cell anemia.

Sickle cell anemia (SCA) is caused by a point mutation in the ß-globin gene that leads to devastating downstream consequences including chronic hemolytic anemia, episodic vascular occlusion, and cumulative organ damage resulting in death. SCA patients show coagulation activation and inflammation even in the absence of vascular occlusion. The coagulation factor fibrinogen is not only central to hemostasis but also plays important roles in pathologic inflammatory processes, in part by engaging neutrophils/macrophages through the αMß2 integrin receptor. To determine whether fibrin(ogen)-mediated inflammation is a driver of SCA-associated pathologies, hematopoietic stem cells from Berkeley sickle mice were transplanted into homozygous Fibγ390-396A mice that express normal levels of a mutant form of fibrin(ogen) that does not engage αMß2 Fibγ390-396A mice with SCA displayed an impressive reduction of reactive oxygen species (ROS) in white blood cells (WBCs), decreased circulating inflammatory cytokines/chemokines, and significantly improved SCA-associated glomerular pathology highlighted by reduced glomerulosclerosis, inflammatory cell infiltration, ischemic lesions, mesangial thickening, mesangial hypercellularity, and glomerular enlargement. In addition, Fibγ390-396A mice with SCA had improved glomerular protective responses and podocyte/mesangial transcriptional signatures that resulted in reduced albuminuria. Interestingly, the fibrinogen γ390-396A mutation had a negligible effect on cardiac, lung, and liver functions and pathologies in the context of SCA over a year-long observation period. Taken together, our data support that fibrinogen significantly contributes to WBC-driven inflammation and ROS production, which is a key driver of SCA-associated glomerulopathy, and may represent a novel therapeutic target against irreversible kidney damage in SCA.

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.

Genetic diminution of circulating prothrombin ameliorates multiorgan pathologies in sickle cell disease mice.

Sickle cell disease (SCD) results in vascular occlusions, chronic hemolytic anemia, and cumulative organ damage. A conspicuous feature of SCD is chronic inflammation and coagulation system activation. Thrombin (factor IIa [FIIa]) is both a central protease in hemostasis and a key modifier of inflammatory processes. To explore the hypothesis that reduced prothrombin (factor II [FII]) levels in SCD will limit vaso-occlusion, vasculopathy, and inflammation, we used 2 strategies to suppress FII in SCD mice. Weekly administration of FII antisense oligonucleotide "gapmer" to Berkeley SCD mice to selectively reduce circulating FII levels to ∼10% of normal for 15 weeks significantly diminished early mortality. More comprehensive, long-term comparative studies were done using mice with genetic diminution of circulating FII. Here, cohorts of FII(lox/-) mice (constitutively carrying ∼10% normal FII) and FII(WT) mice were tracked in parallel for a year following the imposition of SCD via hematopoietic stem cell transplantation. This genetically imposed suppression of FII levels resulted in an impressive reduction in inflammation (reduction in leukocytosis, thrombocytosis, and circulating interleukin-6 levels), reduced endothelial cell dysfunction (reduced endothelial activation and circulating soluble vascular cell adhesion molecule), and a significant improvement in SCD-associated end-organ damage (nephropathy, pulmonary hypertension, pulmonary inflammation, liver function, inflammatory infiltration, and microinfarctions). Notably, all of these benefits were achieved with a relatively modest 1.25-fold increase in prothrombin times, and in the absence of hemorrhagic complications. Taken together, these data establish that prothrombin is a powerful modifier of SCD-induced end-organ damage, and present a novel therapeutic target to ameliorate SCD pathologies.

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.

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.

Genotoxic potential of lineage-specific lentivirus vectors carrying the beta-globin locus control region.

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.

Improved human beta-globin expression from self-inactivating lentiviral vectors carrying the chicken hypersensitive site-4 (cHS4) insulator element.

Effective gene therapy for beta-thalassemia major (beta-TM) requires consistent, high expression of human beta-globin (hbeta-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hbeta/hgamma-globin genes and large elements of the locus control region, resulting in correction of the murine thalassemia intermedia (TI) phenotype and survival of mice with the TM phenotype. However, current LVs show variable hbeta/hgamma-globin expression and require a high number of vector copies/cell for a therapeutic effect. To address this, we designed LVs flanked by the chicken hypersensitive site-4 (cHS4) chromatin insulator element and compared them with their "un-insulated" counterparts. We observed a consistent twofold-higher hbeta expression from insulated vectors in single-copy mouse erythroleukemia cell clones, an increase that resulted from reduced position effect variegation (PEV) and increased probability of expression from individual integrants. This effect was confirmed in vivo: an approximately twofold increase in hbeta expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hbeta-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hbeta-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hbeta expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.

Successful correction of the human Cooley's anemia beta-thalassemia major phenotype using a lentiviral vector flanked by the chicken hypersensitive site 4 chromatin insulator.

beta-Thalassemias are the most common single-gene disorders and are potentially amenable to gene therapy. While retroviral vectors carrying the human beta-globin cassette were notoriously unstable and expressed poorly, considerable progress has now been made using lentiviral vectors (LVs), which stably transmit the beta-globin expression cassette. Mouse studies using LVs have shown correction of the beta-thalassemia-intermedia phenotype and a partial, variable correction of the mouse beta-thalassemia major phenotype, despite the use of beta-globin-hypersensitive sites that are known to result in position-independent effects. Our group used the alpha-globin-hypersensitive site in self-inactivating (SIN) LVs with long-term expression in secondary mice that resisted methylation-associated proviral silencing. However, these vectors also suffered from chromatin position effects. We therefore flanked a SIN-lentiviral vector carrying the human beta-globin expression cassette with a chromatin insulator and studied expression in bone marrow from four patients with transfusion-dependent human thalassemia major. We demonstrated normal levels of human beta-globin expression in erythroid cells produced in in vitro cultures for unilineage erythroid differentiation. There was restoration of effective erythropoiesis and reversal of the abnormally elevated apoptosis that characterizes beta-thalassemia. The gene-corrected human beta-thalassemia progenitor cells were transplanted into immune-deficient mice, where they underwent normal erythroid differentiation, expressed normal levels of human beta-globin, and displayed normal effective erythropoiesis 3-4 months after xenotransplantation. Variability of beta-globin expression in erythroid colonies derived in vitro or from xenograft bone marrow was similar to that seen in normal control subjects. Results show genetic correction of primitive human progenitor cells and normalization of the human thalassemia major phenotype.

Gene Therapy for beta-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 Cooley's 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.