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.

Pediatric tumor cells express erythropoietin and a functional erythropoietin receptor that promotes angiogenesis and tumor cell survival.

Erythropoietin was traditionally considered an erythroid-restricted cytokine, but recent evidence indicates a broader role for it in nonhematopoietic tissues, specifically in neural development. Pediatric solid tumors are mostly developmental in origin, and more than 50% of the solid tumors are neural in origin. We found erythropoietin receptor and erythropoietin expression in common pediatric tumor cells: neuroblastomas, Ewing's sarcoma family of tumors, pediatric brain tumors (medulloblastoma, astrocytoma, and ependymoma), Wilms tumors, rhabdomyosarcomas, and hepatoblastomas (n = 24), and in cell lines derived from some of these tumors (n = 25). Expression of erythropoietin in tumor cell lines was hypoxia-inducible. Addition of exogenous erythropoietin to tumor cell lines expressing erythropoietin receptor increased nuclear DNA binding activity of nuclear factor kappa B and increased the expression of the antiapoptotic genes bcl-1, bcl-xL, and mcl-1. Additionally, exogenous erythropoietin increased production and secretion of angiogenic growth factors, vascular endothelial growth factor, or placenta growth factor from the tumor cell lines, which promoted endothelial cell proliferation and chemotaxis. Erythropoietin receptor expression that promotes tumor cell survival and releases angiogenic growth factors in pediatric tumors has not been previously described. Therefore, a careful evaluation of the impact of erythropoietin is warranted in vivo, in xenograft models of pediatric tumors, followed by evaluation in pediatric patients with cancer.

Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor.

Monocytes from patients with sickle cell disease (SCD) are in an activated state. However, the mechanism of activation of monocytes in SCD is not known. Our studies showed that placenta growth factor (PlGF) activated monocytes and increased mRNA levels of cytokines (tumor necrosis factor-alpha [TNF-alpha] and interleukin-1beta [IL-1beta]) and chemokines (monocyte chemotactic protein-1 [MCP-1], IL-8, and macrophage inflammatory protein-1beta [MIP-1beta]) in both normal monocytes and in the THP-1 monocytic cell line. This increase in mRNA expression of cytochemokines was also reflected in monocytes derived from subjects with SCD. We studied the PlGF-mediated downstream cellular signaling events that caused increased transcription of inflammatory cytochemokines and chemotaxis of THP-1 monocytes. PlGF-mediated cytochemokine mRNA and protein expression was inhibited by PD98059 and wortmannin, inhibitors of mitogen-activated protein kinase kinase (MAPK/MEK) kinase and phosphatidylinositol-3 (PI3) kinase, respectively, but not by SB203580, a p38 kinase inhibitor. PlGF caused a time-dependent transient increase in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2), which was completely inhibited by wortmannin, indicating that activation of PI3 kinase preceded MEK activation. PlGF also induced transient phosphorylation of AKT. MEK and PI3 kinase inhibitors and antibody to Flt-1 abrogated PlGF-induced chemotaxis of THP-1 monocytes. Overexpression of a dominant-negative AKT or a dominant-negative PI3 kinase p85 subunit in THP-1 monocytes attenuated the PlGF-mediated phosphorylation of ERK-1/2, cytochemokine secretion, and chemotaxis. Taken together, these data show that activation of monocytes by PlGF occurs via activation of Flt-1, which results in activation of PI3 kinase/AKT and ERK-1/2 pathways. Therefore, we propose that increased levels of PlGF in circulation play an important role in the inflammation observed in SCD via its effects on monocytes.

Placenta growth factor activates monocytes and correlates with sickle cell disease severity.

Sickle cell disease (SCD) results in chronic hypoxia and secondarily increased erythropoietin concentrations. Leukocytosis and activated monocytes are also observed in SCD in absence of infection or vaso-occlusion (steady state), the reasons for which are unknown. We found that erythroid cells produced placenta growth factor (PlGF), an angiogenic growth factor belonging to the vascular endothelial growth factor (VEGF) family, and its expression was induced in bone marrow CD34+ progenitor cells in the presence of erythropoietin. Furthermore, the steady state circulating PlGF levels in subjects with severe SCD (at least 3 vaso-occlusive crises [VOCs] per year) were 18.5 +/- 1.2 pg/mL (n = 9) compared with 15.5 +/- 1.2 pg/mL (n = 13) in those with mild SCD (fewer than 3 VOCs per year) and 11.3 +/- 0.7 pg/mL (n = 9) in healthy controls (P <.05), suggesting a correlation between PlGF levels and SCD severity. In addition, PlGF significantly increased mRNA levels of the proinflammatory cytochemokines interleukin-1beta, interleukin-8, monocyte chemoattractant protein-1, and VEGF in peripheral blood mononuclear cells (MNCs) of healthy subjects (n = 4; P <.05). Expression of these same cytochemokines was significantly increased in MNCs from subjects with SCD at steady state (n = 14), compared with healthy controls. Of the leukocyte subfractions, PlGF stimulated monocyte chemotaxis (P <.05, n = 3). Taken together, these data show for the first time that erythroid cells intrinsically release a factor that can directly activate monocytes to increase inflammation. The baseline inflammation seen in SCD has always been attributed to sequelae secondary to the sickling phenomenon. We show that PlGF contributes to the inflammation observed in SCD and increases the incidence of vaso-occlusive events.

Collagen-targeted BMP3 fusion proteins arrayed on collagen matrices or porous ceramics impregnated with Type I collagen enhance osteogenesis in a rat cranial defect model.

Bone morphogenetic protein 3 (BMP3) is a potent osteoinductive growth factor belonging to the TGF-beta superfamily. In this study, we engineered a recombinant BMP3 protein to include an auxiliary collagen-targeting domain derived from von Willebrand coagulation factor (vWF). The collagen-targeted BMP3 fusion protein (rhBMP3-C) was expressed in E. coli, purified from bacterial inclusion bodies, renatured under controlled redox conditions, and assayed for biological activity in vitro and in vivo. The renatured rhBMP3-C fusion protein bound tightly to collagen matrices and inhibited DNA synthesis in normal rat calvaria cells and in two out of three human osteosarcoma cell lines tested. Alkaline phosphatase activity was increased in rat calvarial cells and was decreased in osteosarcoma cells in vitro in a dose-dependent manner. Collagen sponges impregnated with rhBMP3-C and implanted subcutaneously in Fischer-344 rats induced dose-dependent dystrophic calcification of the collagen matrix, with no evidence of ectopic bone formation. However, local injection of rhBMP3-C infused in a collagen suspension induced new bone formation on the periosteal surface of rat calvaria. Finally, in a rat cranial defect model, surgical implantation of rhBMP3-C arrayed on either collagen sponges or on porous ceramics coated with Type I collagen exhibited marked osteoinductive properties. Taken together, these results demonstrate the feasibility of engineering and manufacturing targeted-BMPs which exhibit an integral gain-of-function that may be exploited to therapeutic advantage in (i) the enhancement of effective local concentrations, (ii) the prevention of systemic biodistribution and side effects, and (iii) the design of improved osteoinductive matrices.