Conformation-sensitive targeting of lipid nanoparticles for RNA therapeutics.

The successful in vivo implementation of gene expression modulation strategies relies on effective, non-immunogenic delivery vehicles. Lipid nanoparticles are one of the most advanced non-viral clinically approved nucleic-acid delivery systems. Yet lipid nanoparticles accumulate naturally in liver cells upon intravenous administration, and hence, there is an urgent need to enhance uptake by other cell types. Here we use a conformation-sensitive targeting strategy to achieve in vivo gene silencing in a selective subset of leukocytes and show potential therapeutic applications in a murine model of colitis. In particular, by targeting the high-affinity conformation of α4ß7 integrin, which is a hallmark of inflammatory gut-homing leukocytes, we silenced interferon-γ in the gut, resulting in an improved therapeutic outcome in experimental colitis. The lipid nanoparticles did not induce adverse immune activation or liver toxicity. These results suggest that our lipid nanoparticle targeting strategy might be applied for selective delivery of payloads to other conformation-sensitive targets.

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis.

One of the most important molecules for multiple sclerosis pathogenesis is α4 integrin, which is responsible for autoreactive leukocytes migration into the brain. The monoclonal antibody, natalizumab, was introduced to market for blocking the extravasation of autoreactive leukocytes via inhibition of α4 integrin. However, the disadvantages of antibodies provided a suitable background for other agents to be replaced with antibodies. Considering the profound advantages of aptamers over antibodies, aptamer isolation against α4 integrin was intended in the current study. The α4 integrin-specific aptamers were selected using cell-systematic evolution of ligands by exponential enrichment (SELEX) method with human embryonic kidney (HEK)-293T overexpressing α4 integrin and HEK-293T as target and control cells, respectively. Evaluation of selected aptamer was performed through flow cytometric analysis. The selected clones were then sequenced and analyzed for any possible secondary structure and affinity. The results of this study led to isolation of 13 different single-stranded DNA clones in 11 rounds of selection which were categorized to three clusters based on common structural motifs and the equilibrium dissociation constant (K d ) of the most stable structure was calculated. The evaluation of SELEX progress showed growth in aptamer affinity with increasing of the number of cycles. Taken together, the findings of this study demonstrated the isolation of α4-specific single-stranded DNA aptamers with suitable affinity for ligand, which can further be replaced with natalizumab.

Integrin binding site within the gC1q domain orchestrates EMILIN-1-induced lymphangiogenesis.

Lymphatic vessels (LVs) play a pivotal role in the control of tissue homeostasis and also have emerged as important regulators of immunity, inflammation and tumor metastasis. EMILIN-1 is the first ECM protein identified as a structural modulator of the growth and maintenance of LV; accordingly, Emilin1-/- mice display lymphatic morphological alterations leading to functional defects as mild lymphedema, leakage and compromised lymph drainage. Many EMILIN-1 functions are exerted by the binding of its gC1q domain with the E933 residue of α4 and α9ß1 integrins. To investigate the specific regulatory role of this domain on lymphangiogenesis, we generated a transgenic mouse model expressing an E933A-mutated EMILIN-1 (E1-E933A), unable to interact with α4 or α9 integrin. The mutant resulted in abnormal LV architecture with dense, tortuous and irregular networks; moreover, the number of anchoring filaments was reduced and collector valves had aberrant narrowed structures. E933A mutation also affected lymphatic function in lymphangiography assays and made the transgenic mice more prone to lymph node metastases. The finding that the gC1q/integrin interaction is crucial for a correct lymphangiogenesis response was confirmed and reinforced by functional in vitro tubulogenesis assays. In addition, ex vivo thoracic-duct ring assays revealed that E1-E933A-derived lymphatic endothelial cells had a severe reduction in sprouting capacity and were unable to organize into capillary-like structures. All these data provide evidence that the novel "regulatory structural" role of EMILIN-1 in the lymphangiogenic process is played by the integrin binding site within its gC1q domain.

Identification of Peptide Mimotope Ligands for Natalizumab.

Mimotope peptides selected from combinatorial peptide libraries can be used as capture reagents for immunoassay detection of therapeutic monoclonal antibodies (mAbs). We report the use of phage display libraries to identify peptide ligands (VeritopesTM) that bind natalizumab, a therapeutic mAb indicated for use in multiple sclerosis. PKNPSKF is identified as a novel natalizumab-binding motif, and peptides containing this motif demonstrated utility as capture reagents in enzyme-linked immunosorbent assays (ELISAs). A peptide containing the identified motif was shown to be competitive with the natural ligand (α4-integrin) and a neutralizing anti-idiotype antibody for natalizumab binding, indicating that VeritopesTM act as surrogate ligands that bind the antigen binding site of natalizumab. Affinity maturation further confirmed the motif sequence and yielded peptides with greater apparent affinity by ELISA. VeritopesTM are promising assay reagents for therapeutic drug level monitoring.

Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2.

Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible ß-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through ß subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-µ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.

Osteopontin binding to the alpha 4 integrin requires highest affinity integrin conformation, but is independent of post-translational modifications of osteopontin.

Osteopontin (OPN) is a ligand for the α4ß1 integrin, but the physiological importance of this binding is not well understood. Here, we have assessed the effect of post-translational modifications on OPN binding to the α4 integrin on cultured human leukocyte cell lines and compared OPN interaction with α4 integrin to that of VCAM and fibronectin. Jurkat cells, whose α4 integrins are inherently activated, adhered to different preparations of OPN in the presence of Mn(2+): the EC50 of adhesion was not affected by phosphorylation or glycosylation status. Thrombin cleavage of OPN at the C-terminus of the α4 integrin-binding site also did not affect binding affinity. THP-1 cells express a low-affinity conformation of the integrin and adhered to OPN only in the presence of Mn(2+) plus PMA or an activating antibody. This was in contrast to VCAM and fibronectin: THP-1 cells adhered to these ligands without integrin activation. Studies with ligand-induced binding site antibodies demonstrated that the SVVYGLR peptide of OPN bound to the α4 integrin with a similar affinity as the LDV peptide of fibronectin, suggesting that a high off-rate is responsible for the reduced binding of OPN to the low-affinity forms of this integrin. Together, the results suggest OPN has very low affinity for the α4 integrin on human leukocytes under physiological conditions.

CD49d antisense drug ATL1102 reduces disease activity in patients with relapsing-remitting MS.

OBJECTIVE: This study evaluated the efficacy and safety of ATL1102, an antisense oligonucleotide that selectively targets the RNA for human CD49d, the α subunit of very late antigen 4, in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: In a multicenter, double-blind, placebo-controlled randomized phase II trial, 77 patients with RRMS were treated with 200 mg of ATL1102 subcutaneously injected 3 times in the first week and twice weekly for 7 weeks or placebo and monitored for a further 8 weeks. MRI scans were taken at baseline and weeks 4, 8, 12, and 16. The primary endpoint was the cumulative number of new active lesions (either new gadolinium-enhancing T1 lesions or nonenhancing new or enlarging T2 lesions) at weeks 4, 8, and 12. RESULTS: A total of 72 patients completed the study and 74 intention-to-treat patients were assessed. ATL1102 significantly reduced the cumulative number of new active lesions by 54.4% compared to placebo (mean 3.0 [SD 6.12] vs 6.2 [9.89], p = 0.01). The cumulative number of new gadolinium-enhancing T1 lesions was reduced by 67.9% compared to placebo (p = 0.002). Treatment-emergent adverse events included mild to moderate injection site erythema and decrease in platelet counts that returned to within the normal range after dosing. CONCLUSIONS: In patients with RRMS, ATL1102 significantly reduced disease activity after 8 weeks of treatment and was generally well-tolerated. This trial provides evidence for the first time that antisense oligonucleotides may be used as a therapeutic approach in neuroimmunologic disorders. CLASSIFICATION: This study provides Class I evidence that for patients with RRMS, the antisense oligonucleotide ATL1102 reduces the number of new active head MRI lesions.

The hydrophobic contacts between the center of the ßI domain and the α1/α7 helices are crucial for the low-affinity state of integrin α4 ß7.

Integrin α4 ß7 mediates both rolling and firm adhesion of lymphocytes by modulating its affinity to the ligand: mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Integrin activation is associated with allosteric reshaping in the ß subunit I (ßI) domain. A prominently conformational change comprises displacement of the α1 and α7 helices in the ßI domain, suggesting that the location of these helices is important for the change in integrin affinity. In the present study, we report that the hydrophobic contacts between the center of the ß7 I domain and the α1/α7 helices play critical roles in keeping α4 ß7 in a low-affinity state. Using molecular dynamics simulation, we identified nine hydrophobic residues that might be involved in the critical hydrophobic contacts maintaining integrin in a low-affinity state. Integrin ß7 I domain exhibited a lower binding free energy for ligand after disrupting these hydrophobic contacts by substituting the hydrophobic residues with Ala. Moreover, these α4 ß7 mutants not only showed high-affinity binding to soluble MAdCAM-1, but also demonstrated firm cell adhesion to immobilized MAdCAM-1 in shear flow and enhanced the strength of the α4 ß7 -MAdCAM-1 interaction. Disruption of the hydrophobic contacts also induced the active conformation of α4 ß7 . Thus, the findings obtained in the present study reveal an important structural basis for the low-affinity state of integrin.

The novel α4B murine α4 integrin protein splicing variant inhibits α4 protein-dependent cell adhesion.

Integrins affect the motility of multiple cell types to control cell survival, growth, or differentiation, which are mediated by cell-cell and cell-extracellular matrix interactions. We reported previously that the α9 integrin splicing variant, SFα9, promotes WT α9 integrin-dependent adhesion. In this study, we introduced a new murine α4 integrin splicing variant, α4B, which has a novel short cytoplasmic tail. In inflamed tissues, the expression of α4B, as well as WT α4 integrin, was up-regulated. Cells expressing α4B specifically bound to VCAM-1 but not other α4 integrin ligands, such as fibronectin CS1 or osteopontin. The binding of cells expressing WT α4 integrin to α4 integrin ligands is inhibited by coexpression of α4B. Knockdown of α4B in metastatic melanoma cell lines results in a significant increase in lung metastasis. Expression levels of WT α4 integrin are unaltered by α4B, with α4B acting as a regulatory subunit for WT α4 integrin by a dominant-negative effect or inhibiting α4 integrin activation.

How natalizumab binds and antagonizes α4 integrins.

Natalizumab antibody to α4-integrins is used in therapy of multiple sclerosis and Crohn's disease. A crystal structure of the Fab bound to an α4 integrin ß-propeller and thigh domain fragment shows that natalizumab recognizes human-mouse differences on the circumference of the ß-propeller domain. The epitope is adjacent to but outside of a ligand-binding groove formed at the interface with the ß-subunit ßI domain and shows no difference in structure when bound to Fab. Competition between Fab and the ligand vascular cell adhesion molecule (VCAM) for binding to cell surface α4ß1 shows noncompetitive antagonism. In agreement, VCAM docking models suggest that binding of domain 1 of VCAM to α4-integrins is unimpeded by the Fab, and that bound Fab requires a change in orientation between domains 1 and 2 of VCAM for binding to α4ß1. Mapping of species-specific differences onto α4ß1 and α4ß7 shows that their ligand-binding sites are highly conserved. Skewing away from these conserved regions of the epitopes recognized by current therapeutic function-blocking antibodies has resulted in previously unanticipated mechanisms of action.

The membrane-proximal KXGFFKR motif of α-integrin mediates chemoresistance.

Cell adhesion-mediated drug resistance contributes to minimal residual disease and relapse in hematological malignancies. Here, we show that adhesion of Jurkat T-acute lymphoblastic leukemia cells to substrates engaging α4ß1-integrin or α5ß1-integrin promotes chemoresistance to doxorubicin-induced apoptosis. Reconstituted expression of α4δ, a truncated α4-integrin with KXGFFKR as the cytoplasmic motif, in α4-deficient cells promoted chemoresistance to doxorubicin in a manner independent of α4-mediated adhesion. The adhesion-independent chemoresistance did not require ß1-integrin as the heterodimeric pair, since expression of Tacδ, a monomeric nonintegrin transmembrane protein fused to the juxtamembrane KXGFFKR, was sufficient to reproduce the phenomenon. The requirement for integrin-mediated adhesion in stimulation of Akt phosphorylation and activation was bypassed for cells expressing α4δ and Tacδ. Cells expressing α4δ and Tacδ exhibited a high influx of extracellular Ca(2+), and inhibition of Ca(2+) channels with verapamil attenuated the adhesion-independent chemoresistance. Tacδ cells also exhibited greater rates of drug efflux. α4δ and Tacδ interacted with the Ca(2+)-binding protein calreticulin, in a manner dependent on the KXGFFKR motif. Adhesion-mediated engagement of α4-integrins promoted an increased calreticulin-α4 association and greater influx of extracellular Ca(2+) than in nonadherent cells. The α-integrin KXGFFKR motif is involved in adhesion-mediated control of chemoresistance in T cells.

Efficacy and safety of natalizumab in Crohn's disease patients treated at 6 Boston academic hospitals.

BACKGROUND: Despite trials demonstrating its efficacy, many physicians harbor concerns regarding the use of natalizumab in the treatment of patients with refractory Crohn's disease (CD). The purpose of this study was to perform a descriptive analysis of a series of CD patients not currently enrolled in a clinical trial. METHODS: A retrospective case review of patients treated with natalizumab at 6 sites in Massachusetts: Boston Medical Center, Beth Israel Deaconess Medical Center, Brigham & Women's Hospital, Lahey Clinic, Massachusetts General Hospital, and UMass Medical Center. RESULTS: Data on 69 CD patients on natalizumab were collected. At the start of treatment, patients' disease duration was 12 years. A high proportion of patients were women (68%), presented with perianal disease (65%) and upper gastrointestinal tract involvement (14%). Prior nonbiologic therapies were steroids (96%), thiopurines (94%), antibiotics (74%), methotrexate (58%), and at least two anti-tumor necrosis factor agent failures (81%). Sixty-nine percent (44 of 64 patients) with available medical evaluation had a partial or complete clinical response. Loss of response was 13% after an average of 1 year of treatment. Adverse events were infusion reactions, headaches, fever, and infections. No case of progressive multifocal leukoencephalopathy was observed. CONCLUSIONS: In our clinical experience outside the context of a clinical trial, natalizumab is largely reserved for CD patients with extensive ileocolonic disease who have failed conventional immunosuppressants and of at least 2 anti-tumor necrosis factor agents. This drug is, however, well tolerated and offers significant clinical improvement for more than a year in one-third of these difficult-to-treat CD patients.

Natalizumab: a review of its use in the management of relapsing-remitting multiple sclerosis.

Natalizumab (Tysabri®) is a humanized monoclonal antibody against the α4 chain of integrins and was the first targeted therapy to be approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). Natalizumab acts as a selective adhesion molecule antagonist, which binds very late antigen (VLA)-4 and inhibits the translocation of activated VLA-4-expressing leukocytes across the blood-brain barrier into the CNS. In a pivotal phase III clinical trial, natalizumab 300 mg intravenously every 4 weeks for 2 years in adults with RRMS significantly reduced the annualized relapse rate and the risk of sustained progression of disability compared with placebo, as well as significantly increasing the proportion of relapse-free patients at 1 and 2 years. Natalizumab also significantly reduced the number of T2-hyperintense, gadolinium-enhancing and T1-hypointense lesions on magnetic resonance imaging, and significantly reduced the volume of T2-hyperintense and T1-hypointense lesions compared with placebo. Natalizumab recipients generally experienced improved health-related quality of life at 1-2 years. Natalizumab was generally well tolerated in pivotal trials. The only adverse events that were more frequent with natalizumab monotherapy than with placebo were fatigue and allergic reactions. The main safety and tolerability issue with natalizumab is the incidence of progressive multifocal leukoencephalopathy (PML). As long as the risk of PML is managed effectively, natalizumab is a valuable therapeutic option for adults with highly active relapsing forms of multiple sclerosis.

Disruption of disulfide restriction at integrin knees induces activation and ligand-independent signaling of α4ß7.

Control of integrin activation and signaling plays crucial roles in cell adhesion, spreading and migration. Here, we report that selective breakage of two conserved disulfide bonds located at the knees of integrin α4C589-C594 and ß7C494-C526 activated α4ß7. This activated integrin had a unique structure that was different from the typical extended conformation of active integrin. In addition, these activated α4ß7 integrins spontaneously clustered on the cell membrane and triggered integrin downstream signaling independent of ligand binding. Although these disulfide bonds were not broken during α4ß7 activation by inside-out signaling or Mn(2+), they could be specifically reduced by 0.1 mM dithiothreitol, a reducing strength that could be produced in vivo under certain conditions. Our findings reveal a novel mechanism of integrin activation under specific reducing conditions by which integrin can signal and promote cell spreading in the absence of ligand.

Distinct signaling mechanisms regulate migration in unconfined versus confined spaces.

Using a microchannel assay, we demonstrate that cells adopt distinct signaling strategies to modulate cell migration in different physical microenvironments. We studied α4ß1 integrin-mediated signaling, which regulates cell migration pertinent to embryonic development, leukocyte trafficking, and melanoma invasion. We show that α4ß1 integrin promotes cell migration through both unconfined and confined spaces. However, unlike unconfined (2D) migration, which depends on enhanced Rac1 activity achieved by preventing α4/paxillin binding, confined migration requires myosin II-driven contractility, which is increased when Rac1 is inhibited by α4/paxillin binding. This Rac1-myosin II cross talk mechanism also controls migration of fibroblast-like cells lacking α4ß1 integrin, in which Rac1 and myosin II modulate unconfined and confined migration, respectively. We further demonstrate the distinct roles of myosin II isoforms, MIIA and MIIB, which are primarily required for confined and unconfined migration, respectively. This work provides a paradigm for the plasticity of cells migrating through different physical microenvironments.

Teduglutide in Crohn's disease.

INTRODUCTION: The currently available medications for treatment of Crohn's disease (CD) include aminosalicylates, corticosteroids, antibiotics, immunomodulators and biologic agents (infliximab, certolizumab pegol, adalimumab and natalizumab). These agents target the immune and inflammatory pathways of CD, while there is a shortage of agents that target the barrier functions of the gut that are impaired in CD. Glucagon-like peptide 2 is an enterogastrone with strong trophic effects on the intestinal mucosa. Teduglutide , the analog of glucagon-like peptide has been already approved by the US Food and Drug Administration as a treatment of short bowel syndrome. This review discusses the potential use of teduglutide in patients with CD. AREAS COVERED: As there has been only one randomized placebo controlled trial of teduglutide in CD, there is a shortage of data regarding the efficacy of this agent in CD. The literature search was performed using Medline database with the use of the following key words: teduglutide, glucagon-like peptide-2, CD and inflammatory bowel disease. EXPERT OPINION: Based on available data, it can be concluded that this agent seems to be a promising medication in CD and further trials are required to define the place of teduglutide in treatment of CD.

PEG conjugates of potent α4 integrin inhibitors, maintaining sustained levels and bioactivity in vivo, following subcutaneous administration.

Mitsunobu reactions were employed to link t-butyl esters of α4 integrin inhibitors at each of the termini of a three-arm, 40 kDa, branched PEG. Cleavage of the t-butyl esters using HCO2H provided easily isolated PEG derivatives, which are potent α4 integrin inhibitors, and which achieve sustained levels and bioactivity in vivo, following subcutaneous administration to rats.

Characterization of 14-3-3-ζ Interactions with integrin tails.

Integrins are a family of heterodimeric (α+ß) adhesion receptors that play key roles in many cellular processes. Integrins are unusual in that their functions can be modulated from both outside and inside the cell. Inside-out signaling is mediated by binding adaptor proteins to the flexible cytoplasmic tails of the α- and ß-integrin subunits. Talin is one well-known intracellular activator, but various other adaptors bind to integrin tails, including 14-3-3-ζ, a member of the 14-3-3 family of dimeric proteins that have a preference for binding phosphorylated sequence motifs. Phosphorylation of a threonine in the ß2 integrin tail has been shown to modulate ß2/14-3-3-ζ interactions, and recently, the α4 integrin tail was reported to bind to 14-3-3-ζ and associate with paxillin in a ternary complex that is regulated by serine phosphorylation. Here, we use a range of biophysical techniques to characterize interactions between 14-3-3-ζ and the cytoplasmic tails of α4, ß1, ß2 and ß3 integrins. The X-ray structure of the 14-3-3-ζ/α4 complex indicates a canonical binding mode for the α4 phospho-peptide, but unexpected features are also observed: residues outside the consensus 14-3-3-ζ binding motif are shown to be essential for an efficient interaction; in contrast, a short ß2 phospho-peptide is sufficient for high-affinity binding to 14-3-3-ζ. In addition, we report novel 14-3-3-ζ/integrin tail interactions that are independent of phosphorylation. Of the integrin tails studied, the strongest interaction with 14-3-3-ζ is observed for the ß1A variant. In summary, new insights about 14-3-3-ζ/integrin tail interactions that have implications for the role of these molecular associations in cells are described.

NMR structure of integrin α4 cytosolic tail and its interactions with paxillin.

BACKGROUND: Integrins are a group of transmembrane signaling proteins that are important in biological processes such as cell adhesion, proliferation and migration. Integrins are α/ß hetero-dimers and there are 24 different integrins formed by specific combinations of 18 α and 8 ß subunits in humans. Generally, each of these subunits has a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). CTs of integrins are important in bidirectional signal transduction and they associate with a large number of intracellular proteins. PRINCIPAL FINDINGS: Using NMR spectroscopy, we determined the 3-D structure of the full-length α4 CT (Lys968-Asp999) and characterize its interactions with the adaptor protein paxillin. The α4 CT assumes an overall helical structure with a kink in its membrane proximal region. Residues Gln981-Asn997 formed a continuous helical conformation that may be sustained by potential ionic and/or hydrogen bond interactions and packing of aromatic-aliphatic side-chains. ¹5N-¹H HSQC NMR experiments reveal interactions of the α4 CT C-terminal region with a fragment of paxillin (residues G139-K277) that encompassed LD2-LD4 repeats. Residues of these LD repeats including their adjoining linkers showed α4 CT binding-induced chemical shift changes. Furthermore, NMR studies using LD-containing peptides showed predominant interactions between LD3 and LD4 of paxillin and α4 CT. Docked structures of the α4 CT with these LD repeats suggest possible polar and/or salt-bridge and non-polar packing interactions. SIGNIFICANCE: The current study provides molecular insights into the structural diversity of α CTs of integrins and interactions of integrin α4 CT with the adaptor protein paxillin.