Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.

Latent transforming growth factor β-binding protein 1 (LTBP-1) targets latent complexes of transforming growth factor β to the extracellular matrix, where the latent cytokine is subsequently activated by several different mechanisms. Fibrillins are extracellular matrix macromolecules whose primary function is architectural: fibrillins assemble into ultrastructurally distinct microfibrils that are ubiquitous in the connective tissue space. LTBPs and fibrillins are highly homologous molecules, and colocalization in the matrix of cultured cells has been reported. To address whether LTBP-1 functions architecturally like fibrillins, microfibrils were extracted from tissues and analyzed immunochemically. In addition, binding studies were conducted to determine whether LTBP-1 interacts with fibrillins. LTBP-1 was not detected in extracted beaded-string microfibrils, suggesting that LTBP-1 is not an integral structural component of microfibrils. However, binding studies demonstrated interactions between LTBP-1 and fibrillins. The binding site was within three domains of the LTBP-1 C terminus, and in fibrillin-1 the site was defined within four domains near the N terminus. Immunolocalization data were consistent with the hypothesis that LTBP-1 is a fibrillin-associated protein present in certain tissues but not in others. In tissues where LTBP-1 is not expressed, LTBP-4 may substitute for LTBP-1, because the C-terminal end of LTBP-4 binds equally well to fibrillin. A model depicting the relationship between LTBP-1 and fibrillin microfibrils is proposed.

Tumor-infiltrating myeloid cells, including tumor-associated macrophages (TAMs), have been implicated in tumor progression. We recently described a lineage of mouse monocytes characterized by expression of the Tie2 angiopoietin receptor and required for the vascularization and growth of several tumor models. Here, we report that TIE2 expression in human blood identifies a subset of monocytes distinct from classical inflammatory monocytes and comprised within the less abundant “resident” population. These TIE2-expressing monocytes (TEMs) accounted for 2% to 7% of blood mononuclear cells in healthy donors and were distinct from rare circulating endothelial cells and progenitors. In human cancer patients, TEMs were observed in the blood and, intriguingly, within the tumors, where they represented the main monocyte population distinct from TAMs. Conversely, TEMs were hardly detected in nonneoplastic tissues. In vitro, TEMs migrated toward angiopoietin-2, a TIE2 ligand released by activated endothelial cells and angiogenic vessels, suggesting a homing mechanism for TEMs to tumors. Purified human TEMs, but not TEM-depleted monocytes, markedly promoted angiogenesis in xenotransplanted human tumors, suggesting a potentially critical role of TEMs in human cancer progression. Human TEMs may provide a novel, biologically relevant marker of angiogenesis and represent a previously unrecognized target of cancer therapy.

Latent transforming growth factor-: Structural features and mechanisms of activation. Transforming growth factors- are cytokines with a wide range of biological effects. They play a pathologic role in inflammatory and fibrosing diseases such as nephrosclerosis. TGFs are secreted in a latent form due to noncovalent association with latency associated peptide (LAP), which is a homodimer formed from the propeptide region of TGF-. LAP is disulfide linked to another protein, latent TGF- binding protein (LTBP). LTBP has features in common with extracellular matrix proteins, and targets latent TGF- to the matrix. Activation of latent TGF- can be accomplished in vitro by denaturing treatments, plasmin digestion, ionizing radiation and interaction with thrombospondin. The mechanisms by which latent TGF- is activated physiologically are not well understood. Results to date suggest an important role for proteases, particularly plasmin, although other mechanisms probably exist. A general model of activation is proposed in which latent TGF- is released from the extracellular matrix by proteases, localized to cell surfaces, and activated by cell-associated plasmin.

The urokinase‐type plasminogen activator (uPA) and the matrix‐degrading metalloproteinases MMP‐2 and MMP‐9 (type IV collagenases/gelatinases) have been implicated in a variety of invasive processes, including tumor invasion, metastasis and angiogenesis. MMP‐2 and MMP‐9 are secreted in the form of inactive zymogens that are activated extracellularly, a fundamental process for the control of their activity. The physiological mechanism(s) of gelatinase activation are still poorly understood; their comprehension may provide tools to control cell invasion. The data reported in this paper show multiple roles of the uPA–plasmin system in the control of gelatinase activity: (i) both gelatinases are associated with the cell surface; binding of uPA and plasmin(ogen) to the cell surface results in gelatinase activation without the action of other metallo‐ or acid proteinases; (ii) inhibition of uPA or plasminogen binding to the cell surface blocks gelatinase activation; (iii) in soluble phase plasmin degrades both gelatinases; and (iv) gelatinase activation and degradation occur in a dose‐ and time‐dependent manner in the presence of physiological plasminogen and uPA concentrations. Thus, the uPA–plasmin system may represent a physiological mechanism for the control of gelatinase activity.