Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential.

Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in "enhancerless" self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.

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.

Activated Transcription Factor 3 in Association with Histone Deacetylase 6 Negatively Regulates MicroRNA 199a2 Transcription by Chromatin Remodeling and Reduces Endothelin-1 Expression.

Previous studies showed that high levels of placenta growth factor (PlGF) correlated with increased plasma levels of endothelin-1 (ET-1), a potent vasoconstrictor, in sickle cell disease (SCD). PlGF-mediated transcription of the ET-1 gene occurs by activation of hypoxia inducible factor 1α (HIF-1α) and posttranscriptionally by microRNA 199a2 (miR-199a2), which targets the 3' untranslated region (UTR) of HIF-1α mRNA. However, relatively less is known about how PlGF represses the expression of miR-199a2 located in the DNM3 opposite strand (DNM3os) transcription unit. Here, we show that PlGF induces the expression of activated transcription factor 3 (ATF3), which, in association with accessory proteins (c-Jun dimerization protein 2 [JDP2], ATF2, and histone deacetylase 6 [HDAC6]), as determined by proteomic analysis, binds to the DNM3os promoter. Furthermore, we show that association of HDAC6 with ATF3 at its binding site in this promoter was correlated with repression of miR-199a2 transcription, as shown by DNM3os transcription reporter and chromatin immunoprecipitation (ChIP) assays. Tubacin, an inhibitor of HDAC6, antagonized PlGF-mediated repression of DNM3os/pre-miR-199a2 transcription with a concomitant reduction in ET-1 levels in cultured endothelial cells. Analysis of lung tissues from Berkeley sickle (BK-SS) mice showed increased levels of ATF3 and increased expression of ET-1. Delivery of tubacin to BK-SS mice significantly attenuated plasma ET-1 and PlGF levels. Our studies demonstrated that ATF3 in conjunction with HDAC6 acts as a transcriptional repressor of the DNM3os/miR-199a2 locus.

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.

Vasculopathy-associated hyperangiotensinemia mobilizes haematopoietic stem cells/progenitors through endothelial AT2R and cytoskeletal dysregulation.

Patients with organ failure of vascular origin have increased circulating haematopoietic stem cells and progenitors (HSC/P). Plasma levels of angiotensin II (Ang-II), are commonly increased in vasculopathies. Hyperangiotensinemia results in activation of a very distinct Ang-II receptor set, Rho family GTPase members, and actin in bone marrow endothelial cells (BMEC) and HSC/P, which results in decreased membrane integrin activation in both BMEC and HSC/P, and in HSC/P de-adhesion and mobilization. The Ang-II effect can be reversed pharmacologically and genetically by inhibiting Ang-II production or signalling through BMEC AT2R, HSCP Ang-II receptor type 1 (AT1R)/AT2R or HSC/P RhoA, but not by interfering with other vascular tone mediators. Hyperangiotensinemia and high counts of circulating HSC/P seen in sickle cell disease (SCD) as a result of vascular damage, is significantly decreased by Ang-II inhibitors. Our data define for the first time the role of Ang-II HSC/P traffic regulation and redefine the haematopoietic consequences of anti-angiotensin therapy in SCD.