Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

Endothelial CD47 promotes vascular endothelial-cadherin tyrosine phosphorylation and participates in T cell recruitment at sites of inflammation in vivo.

At sites of inflammation, endothelial adhesion molecules bind leukocytes and transmit signals required for transendothelial migration (TEM). We previously reported that adhesive interactions between endothelial cell CD47 and leukocyte signal regulatory protein γ (SIRPγ) regulate human T cell TEM. The role of endothelial CD47 in T cell TEM in vivo, however, has not been explored. In this study, CD47⁻/⁻ mice showed reduced recruitment of blood T cells as well as neutrophils and monocytes in a dermal air pouch model of TNF-α-induced inflammation. Reconstitution of CD47⁻/⁻ mice with wild-type bone marrow cells did not restore leukocyte recruitment to the air pouch, indicating a role for endothelial CD47. The defect in leukocyte TEM in the CD47⁻/⁻ endothelium was corroborated by intravital microscopy of inflamed cremaster muscle microcirculation in bone marrow chimera mice. In an in vitro human system, CD47 on both HUVEC and T cells was required for TEM. Although previous studies showed CD47-dependent signaling required G(αi)-coupled pathways, this was not the case for endothelial CD47 because pertussis toxin, which inactivates G(αi), had no inhibitory effect, whereas G(αi) was required by the T cell for TEM. We next investigated the endothelial CD47-dependent signaling events that accompany leukocyte TEM. Ab-induced cross-linking of CD47 revealed robust actin cytoskeleton reorganization and Src- and Pyk-2-kinase dependent tyrosine phosphorylation of the vascular endothelial-cadherin cytoplasmic tail. This signaling was pertussis toxin insensitive, suggesting that endothelial CD47 signaling is independent of G(αi). These findings suggest that engagement of endothelial CD47 by its ligands triggers outside-in signals in endothelium that facilitate leukocyte TEM.

Role of leukemia cell invadosome in extramedullary infiltration.

Acute myelogenous leukemias (AMLs) are characterized by medullary and extramedullary invasion. We hypothesized that a supramolecular complex, the leukemia-cell invadosome, which contains certain integrins, matrix metalloproteinases (MMPs), and other as-yet unidentified proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. Here we show that the specific binding of MMP-9 to leukocyte surface beta(2) integrin is required for pericellular proteolysis and migration of AML-derived cells. An efficient antileukemia effect was obtained by the hexapeptide HFDDDE, a motif of the MMP-9 catalytic domain that mediates integrin binding: HFDDDE prevented proMMP-9 binding, transmigration through a human endothelial cell layer, and extracellular matrix degradation. Notably, the functional protein anchorage between beta(2) integrin and proMMP-9 described in this study does not involve the enzymatic active sites targeted by known MMP inhibitors. Taken together, our results provide a biochemical working definition for the human leukemia invadosome. Disruption of specific protein complexes within this supramolecular target complex may yield a new class of anti-AML drugs with anti-invasion (rather than or in addition to cytotoxic) attributes.

Endothelial CD47 interaction with SIRPgamma is required for human T-cell transendothelial migration under shear flow conditions in vitro.

Leukocyte transendothelial migration (TEM) is a critical event during inflammation. CD47 has been implicated in myeloid cell migration across endothelium and epithelium. CD47 binds to signal regulatory protein (SIRP), SIRPalpha and SIRPgamma. So far, little is known about the role of endothelial CD47 in T-cell TEM in vivo or under flow conditions in vitro. Fluorescence-activated cell sorting and biochemical analysis show that CD3(+) T cells express SIRPgamma but not SIRPalpha, and fluorescence microscopy showed that CD47 was enriched at endothelial junctions. These expression patterns suggested that CD47 plays a role in T-cell TEM through binding interactions with SIRPgamma. We tested, therefore, whether CD47-SIRPgamma interactions affect T-cell transmigration using blocking mAb against CD47 or SIRPgamma in an in vitro flow model. These antibodies inhibited T-cell TEM by 70% plus or minus 6% and 82% plus or minus 1%, respectively, but had no effect on adhesion. In agreement with human mAb studies, transmigration of murine wild-type T helper type 1 cells across TNF-alpha-activated murine CD47(-/-) endothelium was reduced by 75% plus or minus 2% even though murine T cells appear to lack SIRPgamma. Nonetheless, these findings suggest endothelial cell CD47 interacting with T-cell ligands, such as SIRPgamma, play an important role in T-cell transendothelial migration.

Activation of NMDA receptors promotes dendritic spine development through MMP-mediated ICAM-5 cleavage.

Matrix metalloproteinase (MMP)-2 and -9 are pivotal in remodeling many tissues. However, their functions and candidate substrates for brain development are poorly characterized. Intercellular adhesion molecule-5 (ICAM-5; Telencephalin) is a neuronal adhesion molecule that regulates dendritic elongation and spine maturation. We find that ICAM-5 is cleaved from hippocampal neurons when the cells are treated with N-methyl-d-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA). The cleavage is blocked by MMP-2 and -9 inhibitors and small interfering RNAs. Newborn MMP-2- and MMP-9-deficient mice brains contain more full-length ICAM-5 than wild-type mice. NMDA receptor activation disrupts the actin cytoskeletal association of ICAM-5, which promotes its cleavage. ICAM-5 is mainly located in dendritic filopodia and immature thin spines. MMP inhibitors block the NMDA-induced cleavage of ICAM-5 more efficiently in dendritic shafts than in thin spines. ICAM-5 deficiency causes retraction of thin spine heads in response to NMDA stimulation. Soluble ICAM-5 promotes elongation of dendritic filopodia from wild-type neurons, but not from ICAM-5-deficient neurons. Thus, MMPs are important for ICAM-5-mediated dendritic spine development.

alpha-Chain phosphorylation of the human leukocyte CD11b/CD18 (Mac-1) integrin is pivotal for integrin activation to bind ICAMs and leukocyte extravasation.

The promiscuous CD11b/CD18 (Mac-1) integrin has important roles in regulating many immunologic functions such as leukocyte adhesion and emigration from the bloodstream via interactions with the endothelial ligands ICAM-1 and ICAM-2, iC3b-mediated phagocytosis, and apoptosis. However, the mechanisms for Mac-1 inside-out activation have remained poorly understood. Phosphorylation of integrin cytoplasmic domains is emerging as an important mechanism of regulating integrin functions. Here, we have studied phosphorylation of human CD11b, which takes place on the cytoplasmic Ser1126 in neutrophils. We show that mutation of the serine phosphorylation site leads to inability of Mac-1 to become activated to bind the cellular ligands ICAM-1 and ICAM-2. However, CD11b-mutant cells are fully capable of binding other studied CD11b ligands (ie, iC3b and denatured BSA). Activation epitopes expressed in the extracellular domain of the integrin and affinity for soluble ICAM ligands were decreased for the mutated integrin. Additionally, the mutation resulted in inhibition of chemokine-induced migration in a transendothelial assay in vitro and significantly reduced the accumulation of intravenously administered cells in the spleen and lungs of Balb/c mice. These results characterize a novel selective mechanism of Mac-1-integrin activation, which mediates leukocyte emigration from the bloodstream to the tissues.

Cell-surface association between matrix metalloproteinases and integrins: role of the complexes in leukocyte migration and cancer progression.

Leukocyte motility is known to be dependent on both beta2-integrins and matrix metalloproteinases MMP-2/-9 or gelatinases, which mediate leukocyte adhesion and the proteolysis needed for invasion, respectively. Gelatinases not only play an important role in cell migration, tissue remodeling, and angiogenesis during development, but are also involved in the progression and invasiveness of many cancers, including leukemias. The concept that MMPs associate with integrins, as well as their importance in some physiologic and pathologic conditions, has been advanced previously but has not been examined on leukocytes. This review will examine mainly the function of the MMP-integrin complexes in normal leukocyte migration and the effect of integrin and broad-spectrum MMP inhibitors in tumor progression.

Stabilization of the activated alphaMbeta2 integrin by a small molecule inhibits leukocyte migration and recruitment.

Integrins are potential targets for the development of antiinflammatory agents. Here we develop a novel high-throughput assay by allowing a chemical library to compete with phage display peptide binding and identify a novel small-molecule ligand to the leukocyte-specific alpha(M)beta(2) integrin. The identified thioxothiazolidine-containing compound, IMB-10, had an unexpected activity in that it stabilized binding of alpha(M)beta(2) to its endogenous ligands proMMP-9 and fibrinogen. Single amino acid substitutions in the activity-regulating C-terminal helix and the underlying region in the ligand-binding I domain of the integrin suppressed the effect of IMB-10. A computational model indicated that IMB-10 occupies a distinct cavity present only in the activated form of the integrin I domain. IMB-10 inhibited alpha(M)beta(2)-dependent migration in vitro and inflammation-induced neutrophil emigration in vivo. Stabilization of integrin-mediated adhesion by a small molecule is a novel means to inhibit cell migration and may have a utility in treatment of inflammatory diseases involving leukocyte recruitment.

Peptide-mediated delivery of therapeutic and imaging agents into mammalian cells.

Modern molecular targeting provides new opportunities for imaging, diagnosis and treatment of diseases. Small molecular weight peptides have the potential for enhancing targeting of compounds, and they may also have therapeutic effects by themselves. The limiting step for successful molecular targeting is the development of efficient peptide delivery systems. This review will focus on peptides developed by phage display and combinatorial chemistry for the delivery of pharmaceuticals, radioactive compounds and gene expression vectors. Target cell-specific delivery can be improved by peptides that penetrate the cell membrane or alternatively induce receptor-mediated endocytosis. In addition, peptides that contain endosomal escape signals or nuclear localization motifs may help trafficking of therapeutics to appropriate locations inside the cell. Small molecule radiolabelled peptides are the preferred agents for targeting and for diagnostic imaging of various organs as they are easily synthesized, effectively penetrate tissues, and are rapidly cleared from the circulation. Such peptides have been tested in animals and humans in the fields of cancer, cardiology, neurology, inflammation/infection, atherosclerosis and thrombosis.

Intracellular and cell surface localization of a complex between alphaMbeta2 integrin and promatrix metalloproteinase-9 progelatinase in neutrophils.

We have recently demonstrated that promatrix metalloproteinases (proMMPs), particularly proMMP-9, are potent ligands of the leukocyte beta(2) integrins. We studied here the complex formation between proMMP-9 and alpha(M)beta(2), the major MMP and integrin of neutrophils. On resting neutrophils, the proMMP-9/alpha(M)beta(2) complex was primarily detected in intracellular granules, but after cellular activation it became localized to the cell surface, as demonstrated by immunoprecipitation and double immunofluorescence. Further indication of the complex formation was that neutrophils and alpha(M)beta(2)-transfected L cells, but not the wild-type L cells or leukocyte adhesion deficiency cells, bound to immobilized proMMP-9 or its recombinant catalytic domain in a beta(2) integrin-dependent manner. Peptides that bound to the alpha(M) integrin-I domain and inhibited its complex formation with proMMP-9 prevented neutrophil migration in a transendothelial assay in vitro and in a thioglycolate-elicited peritonitis in vivo. These results suggest that the translocating proMMP-9/alpha(M)beta(2) complex may be part of the cell surface machinery guiding neutrophil migration.

Identification of a negatively charged peptide motif within the catalytic domain of progelatinases that mediates binding to leukocyte beta 2 integrins.

The alpha M beta 2 integrin of leukocytes can bind a variety of ligands. We screened phage display libraries to isolate peptides that bind to the alpha M I domain, the principal ligand binding site of the integrin. Only one peptide motif, (D/E)(D/E)(G/L)W, was obtained with this approach despite the known ligand binding promiscuity of the I domain. Interestingly, such negatively charged sequences are present in many known beta 2 integrin ligands and also in the catalytic domain of matrix metalloproteinases (MMPs). We show that purified beta 2 integrins bind to pro-MMP-2 and pro-MMP-9 gelatinases and that that the negatively charged sequence of the MMP catalytic domain is an active beta 2 integrin-binding site. Furthermore, a synthetic DDGW-containing phage display peptide inhibited the ability of beta 2 integrin to bind progelatinases but did not inhibit the binding of cell adhesion-mediating substrates such as intercellular adhesion molecule-1, fibrinogen, or an LLG-containing peptide. Immunoprecipitation and cell surface labeling demonstrated complexes of pro-MMP-9 with both the alpha M beta 2 and alpha L beta 2 integrins in leukocytes, and pro-MMP-9 colocalized with alpha M beta 2 in cell surface protrusions. The DDGW peptide and the gelatinase-specific inhibitor peptide CTTHWGFTLC blocked beta 2 integrin-dependent leukocyte migration in a transwell assay. These results suggest that leukocytes may move in a progelatinase-beta 2 integrin complex-dependent manner.

Characterization of ICAM-4 binding to the I domains of the CD11a/CD18 and CD11b/CD18 leukocyte integrins.

Intercellular adhesion molecule-4 (ICAM-4, LW blood group antigen), a member of the immunoglobulin superfamily expressed on red cells, has been reported to bind to CD11a/CD18 and CD11b/CD18 leukocyte integrins. The location of the ICAM-4 binding sites on CD11a/CD18 and CD11b/CD18 are not known. CD11/CD18 integrin I domains have been found to act as major binding sites for physiological ligands and a negatively charged glutamic acid in ICAMs is considered important for binding. ICAM-4 lacks such a residue, which is replaced by an arginine. However, we demonstrate here that ICAM-4 in red cells and transfected fibroblasts interacts specifically with the I domains of CD11a/CD18 and CD11b/CD18 integrins. The binding was inhibited by anti-I domain and anti-ICAM-4 antibodies and it was dependent on divalent cations. Interestingly, ICAM-4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearly reduced. Using a solid phase assay, we were able to show that isolated I domains directly and specifically bind to purified recombinant ICAM-4 in a cation dependent manner. Competition experiments indicated that the binding sites in ICAM-4 for the CD11a and CD11b I domains are different. However, the ICAM-4 binding region in both I domains seems to overlap with the regions recognized by the ICAM-1 and ICAM-2. Thus we have established that the I domains contain an ICAM-4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins.