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Biomolecule misfolding and aggregation play a major role in human disease, spanning from neurodegeneration to cancer. Inhibition of these processes is of considerable interest, and due to the multifactorial nature of these diseases, the development of drugs that act on multiple pathways simultaneously is a promising approach. This Feature Article focuses on the development of multifunctional molecules designed to inhibit the misfolding and aggregation of the amyloid-β (Aβ) peptide in Alzheimer's disease (AD), and the mutant p53 protein in cancer. While for the former, the goal is to accelerate the removal of the Aβ peptide and associated aggregates, for the latter, the goal is reactivation via stabilization of the active folded form of mutant p53 protein and/or aggregation inhibition. Due to the similar aggregation pathway of the Aβ peptide and mutant p53 protein, a common therapeutic approach may be applicable.

Extracellular vesicles (EVs) contain more than 100 proteins. Whether there are EVs proteins that act as an 'organiser' of protein networks to generate a new or different biological effect from that identified in EV-producing cells has never been demonstrated. Here, as a proof-of-concept, we demonstrate that EV-G12D-mutant KRAS serves as a leader that forms a protein complex and promotes lung inflammation and tumour growth via the Fn1/IL-17A/FGF21 axis. Mechanistically, in contrast to cytosol derived G12D-mutant KRAS complex from EVs-producing cells, EV-G12D-mutant KRAS interacts with a group of extracellular vesicular factors via fibronectin-1 (Fn1), which drives the activation of the IL-17A/FGF21 inflammation pathway in EV recipient cells. We show that: (i), depletion of EV-Fn1 leads to a reduction of a number of inflammatory cytokines including IL-17A; (ii) induction of IL-17A promotes lung inflammation, which in turn leads to IL-17A mediated induction of FGF21 in the lung; and (iii) EV-G12D-mutant KRAS complex mediated lung inflammation is abrogated in IL-17 receptor KO mice. These findings establish a new concept in EV function with potential implications for novel therapeutic interventions in EV-mediated disease processes., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

In this review, we shed light on recent advances regarding the characterization of biochemical pathways of cellular mechanosensing and mechanotransduction with particular attention to their role in neurodegenerative disease pathogenesis. While the mechanistic components of these pathways are mostly uncovered today, the crosstalk between mechanical forces and soluble intracellular signaling is still not fully elucidated. Here, we recapitulate the general concepts of mechanobiology and the mechanisms that govern the mechanosensing and mechanotransduction processes, and we examine the crosstalk between mechanical stimuli and intracellular biochemical response, highlighting their effect on cellular organelles' homeostasis and dysfunction. In particular, we discuss the current knowledge about the translation of mechanosignaling into biochemical signaling, focusing on those diseases that encompass metabolic accumulation of mutant proteins and have as primary characteristics the formation of pathological intracellular aggregates, such as Alzheimer's Disease, Huntington's Disease, Amyotrophic Lateral Sclerosis and Parkinson's Disease. Overall, recent findings elucidate how mechanosensing and mechanotransduction pathways may be crucial to understand the pathogenic mechanisms underlying neurodegenerative diseases and emphasize the importance of these pathways for identifying potential therapeutic targets.

Microbial proteins have recently been found to have more benefits in clinical disease treatment because of their better-developed strategy and properties than traditional medicine. In this study, we investigated the effectiveness of a truncated peptide synthesized from the C-terminal sequence of pneumolysin, i.e., C70PLY4, in Streptococcus pneumoniae , in treating chronic inflammatory conditions. It has been shown that C70PLY4 significantly blocks the transendothelial migration of neutrophils and attenuates the formation of atherosclerotic plaque and the secretion of soluble forms of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in high-fat-diet/streptozotocin-induced inflammatory rats. The mechanism and the docking simulation analysis further indicated that C70PLY4 might serve as a Toll-like receptor 4 (TLR4) antagonist by competing for the binding site of MD2, an indispensable protein for lipopolysaccharide (LPS)-TLR4 interaction signaling, on the TLR4 structure. Moreover, compared to the full-length PLY, C70PLY4 seems to have no cytotoxicity in human vascular endothelial cells. Our study elucidated a possible therapeutic efficacy of C70PLY4 in reducing chronic inflammatory conditions and clarified the underlying mechanism. Thus, our findings identify a new drug candidate that, by blocking TLR4 activity, could be an effective treatment for patients with chronic inflammatory diseases.

Severe photoreceptor cell death in retinal degenerative diseases leads to partial or complete blindness. Optogenetics is a promising strategy to treat blindness. The feasibility of this strategy has been demonstrated through the ectopic expression of microbial channelrhodopsins (ChRs) and other genetically encoded light sensors in surviving retinal neurons in animal models. A major drawback for ChR-based visual restoration is low light sensitivity. Here, we report the development of highly operational light-sensitive ChRs by optimizing the kinetics of a recently reported ChR variant, Chloromonas oogama (CoChR). In particular, we identified two CoChR mutants, CoChR-L112C and CoChR-H94E/L112C/K264T, with markedly enhanced light sensitivity. The improved light sensitivity of the CoChR mutants was confirmed by ex vivo electrophysiological recordings in the retina. Furthermore, the CoChR mutants restored the vision of a blind mouse model under ambient light conditions with remarkably good contrast sensitivity and visual acuity, as evidenced by the results of behavioral assays. The ability to restore functional vision under normal light conditions with the improved CoChR variants removed a major obstacle for ChR-based optogenetic vision restoration., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Inflammatory bowel disease (IBD) remains a major health challenge due in part to unsafe and limited treatment options, hence there is the need for alternatives. CXCL8/interleukin 8 (IL-8) is elevated in inflammation, and binds preferentially to G protein-couple receptors (GPCRs) CXCR1/2 of the CXC chemokine family to initiate cascades of downstream inflammatory signals. A mutant CXCL8 protein, CXCL8(3-72)K11R/G31P (G31P), competitively and selectively binds to CXCR1/2, making CXCL8 redundant. We explore the therapeutic potential of G31P in dextran sulfate sodium (DSS) induced ulcerative colitis (UC), and the corresponding effect if G31P treatment is augmented with Lactobacillus acidophilus (LACT). The treatment options administered significantly reduced TNF-α, IFN-γ, IL-1β, IL-6, and IL-8, but maintained elevated levels of IL-10. CD68 and F4/80 expressions were down-regulated and showed restricted infiltration to inflamed colon, while IL-17F levels were insignificantly different from the DSS treated mice. Also, we observed up-regulation of IL-17A in G31P + LACT but not G31P treated mice if compared with Control group. The treatments ameliorated colonic fibrosis by reducing VEGF, TGF-β, MMP-2 and MMP-9. In addition, we observed elevated levels of E-cadherin, and marginal up-regulation of occludin, suggesting the role of the treatments in regulating tight intestinal junction and adherence proteins. Mechanism-wise, G31P interferes with AKT and ERK signaling pathways. Our study suggests that G31P confers protection in IBD, particularly UC, and when G31P treatment is augmented with Lactobacillus acidophilus, the protection is variably enhanced., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Fibroblast growth factor 21 (FGF21) is a promising regulator of glucose and lipid metabolism with multiple beneficial effects including hypoglycemic and lipid-lowering. Previous studies have reported that FGF21 is expected to become a new drug for treatment of diabetes. Liraglutide and insulin glargine are the two representative anti-diabetic biological drugs. In the current study, we aim to compare the long-term pharmacological efficacy of mFGF21 (an FGF21 analogue), liraglutide and insulin glargine in type 2 diabetic db/db mice. Db/db mice were initially treated with three kinds of proteins (25nmol/kg/day) by subcutaneous injection once a day for 4weeks, then subsequently be treated with once every two days for next 4weeks. After 8weeks of treatments, the blood glucose levels, body weights, glycosylated hemoglobin levels, fasting insulin levels, serum lipid profiles, hepatic biochemical parameters, oral glucose tolerance tests and hepatic mRNA expression levels of several proteins (GK, G6P, GLUT-1 and GLUT-4) associated with glucose metabolism of the experimental mice were detected. Results demonstrated that three proteins could significantly decrease the fed blood glucose levels of db/db mice. After treatment for 1week, the fed blood glucose levels of db/db mice in liraglutide group were significantly lower than those in mFGF21 and insulin glargine groups. However, after 2weeks of administration, the long-lasting hypoglycemic effect of mFGF21 was superior to liraglutide and insulin glargine up to the end of the experiments. Compared with liraglutide and insulin glargine, mFGF21 significantly reduced the glycosylated hemoglobin levels and improved the ability on glycemic control, insulin resistance, serum lipid and liver function states in db/db mice after 8weeks treatments. In addition, mFGF21 regulated glucose metabolism through increasing the mRNA expression levels of GK and GLUT-1, and decreasing the mRNA expression level of G6P. But liraglutide and insulin glargine could only up-regulate the mRNA expression of GLUT-4. In summary, as a hypoglycemic drug for long-term treatment, mFGF21 has the potential to be an ideal drug candidate for the therapy of type 2 diabetes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The TP53 tumor suppressor is the most frequently mutated gene in human cancers. In recent years, a blooming of research efforts based on both cell lines and mouse models have highlighted how deeply mutant p53 proteins affect fundamental cellular pathways with cancer-promoting outcomes. Neomorphic mutant p53 activities spread over multiple levels, impinging on chromatin structure, transcriptional regulation and microRNA maturation, shaping the proteome and the cell's metabolic pathways, and also exerting cytoplasmic functions and displaying cell-extrinsic effects. These tumorigenic activities are inextricably linked with the blend of highly corrupted processes that characterize the tumor context. Recent studies indicate that successful strategies to extract core aspects of mutant p53 oncogenic potential and to identify unique tumor dependencies entail the superimposition of large-scale analyses performed in multiple experimental systems, together with a mindful use of animal models. This will hopefully soon lead to the long-awaited inclusion of mutant p53 as an actionable target of clinical antitumor therapies., (© 2016 Federation of European Biochemical Societies.)

Acute renal failure, a serious condition characterised by a drastic decline in renal function, often follows ischaemia/reperfusion (I/R) episodes. I/R is characterised by necrosis, inflammation and activation of coagulation, in concert causing renal tissue damage. In this context, activated protein C (APC) might be of importance in the pathogenesis of renal I/R. APC is a serine protease which has anticoagulant but also several anti-inflammatory and cytoprotective effects such as protection of endothelial barrier function. It was our objective to study the role of cytoprotective and anticoagulant functions of APC during renal I/R. C57BL/6j mice subjected to renal I/R were treated with intraperitoneally injected exogenous human APC, or two mutant forms of APC (200 µg/kg) which specifically lack anticoagulant or signalling properties. In a different experiment mice received specific monoclonal antibodies (20 mg/kg) that block the cytoprotective and/or anticoagulant properties of endogenous APC. Treatment with APC reduced tubular injury and enhanced renal function without altering the inflammatory response and did reduce renal fibrin deposition. Administration of APC mutant lacking anticoagulant properties reduced renal damage and enhanced renal function. Blocking the anticoagulant and cytoprotective functions of endogenous APC resulted in elevated tubular damage and reduced tubular cell proliferation, however, without influencing renal function or the inflammatory response. Furthermore, blocking both the anticoagulant and cytoprotective effects of APC resulted in dramatic renal interstitial haemorrhage, indicative of impaired vascular integrity. Blocking only the anticoagulant function of APC did not result in interstitial bleeding. In conclusion, the renoprotective effect of APC during I/R is independent of its anticoagulant properties.

Background: Cytokine fusion protein that modulates the immune response holds great potential for cancer immunotherapy. IL-2 is an effective treatment against advanced cancers. However, the therapeutic efficacy of IL-2 is limited by severe systemic toxicity. Several mutants recombinant IL-2 can increase antitumor activity and minimize systemic toxicity. Melittin is an attractive anticancer candidate because of its wide-spectrum lytic properties. We previously generated a bifunctional fusion protein melittin-MIL-2, composed of melittin and a mutant IL-2. The melittin-MIL-2 inhibited the growth of human ovarian cancer SKOV3 cells in vitro and in vivo tumor growth. However, whether this antitumor effect could also be used in cancer immunotherapy was unknown. To assess its cancer immunotherapy potential, we further investigated its more effective antitumor immune response and antitumor effect against cancers of different tissue origins in vitro and in vivo., Methods: The specific IL-2 activity of the melittin-MIL-2 fusion protein was tested on the cytokine growth dependent cell line CTLL-2. The cytolytic activity was detected by standard 4-h (51)Cr-release assays. PBMC stimulation in response to the melittin-MIL-2 was determined by IFN-γ release assay. We observed the cancer cell proliferation of different tissue origins by MTT assay. The ability of melittin-MIL-2 to inhibit tumor growth in vivo was evaluated by using human liver (SMMC-7721 cancer cells), lung (A549 cancer cells) and ovarian (SKOV3 cancer cells) cancer xenograft models. To assess the immunity within the tumor microenvironment, the level of some cytokines including IFN-γ, TNF-α, IL-12 and IL-4 was analyzed by ELISA. We injected the MDA-MB-231 cells and the melittin-MIL-2 into mice, and the anti-metastatic effect was examined by counting nodules in the lung., Results: The melittin-MIL-2 was more effective in inducing T cell and NK-cell cytotoxicity. The fusion protein significantly increased IFN-γ production in PBMCs. In vitro, the melittin-MIL-2 mediated immune cells killing or directly killed the cancer cell lines of different tissue origins. In vivo, the fusion protein exhibited stronger inhibition on the growth of transplanted human tumors compared to rIL-2. The melittin-MIL-2 treatment promoted the IFN-γ secretion in tumor tissues and decreased the immunosuppressive cells in vivo. Furthermore, the fusion protein reduced lung metastasis of breast cancer., Conclusions: This study provides the evidence that the melittin-MIL-2 can produce stronger immune stimulation and antitumor effects, and the fusion protein is a potent candidate for cancer immunotherapy.

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

Current HIV-1 gene therapy approaches aim at stopping the viral life cycle at its earliest steps, such as entry or immediate postentry events. Among the most widely adopted strategies are CCR5 downregulation/knockout and the use of broadly neutralizing antibodies. However, the long-term efficacy and side effects are still unclear. TRIM5α is an interferon-stimulated restriction factor that can intercept incoming retroviruses within one hour of cytosolic entry and potently inhibit the infectivity of restriction-sensitive viruses. The human TRIM5α (TRIM5αhu) generally does not efficiently target HIV-1, but point mutations in its capsid-binding domain can confer anti-HIV-1 activity. Although the mechanisms by which TRIM5αhu mutants inhibit HIV-1 are relatively well understood, their characterization as potential transgenes for gene therapy is lacking. Additionally, previous reports of general immune activation by overexpression of TRIM5α have hindered its broad adoption as a potential transgene. Here we demonstrate the ability of the R332G-R335G TRIM5αhu mutant to efficiently restrict highly divergent HIV-1 strains, including Group O, as well as clinical isolates bearing cytotoxic T lymphocyte escape mutations. R332G-R335G TRIM5αhu efficiently protected human lymphocytes against HIV-1 infection, even when expressed at relatively low levels following lentiviral transduction. Most importantly, under these conditions Rhesus macaque TRIM5α (TRIM5αRh) and TRIM5αhu (wild-type or mutated) had no major effects on the NF-κB pathway. Transgenic TRIM5α did not modulate the kinetics of IκBα, JunB, and TNFAIP3 expression following TNF-α treatment. Finally, we show that human lymphocytes expressing R332G-R335G TRIM5αhu have clear survival advantages over unmodified parental cells in the presence of pathogenic, replication-competent HIV-1. These results support the relevance of R332G-R335G and other mutants of TRIM5αhu as candidate effectors for HIV-1 gene therapy.

There is a clinical need to develop safe therapeutic strategies to mitigate bleeding. Previously, we found that a novel zymogen-like factor Xa variant (FXa-I16L) was effective in correcting the coagulation defect in hemophilic mice. Here we expand the mutational framework to tune the FX(a) zymogen-like state. Alteration of FXa zymogenicity yields variants (V17M, I16L, I16M, V17T, V17S, and I16T) with a wide range (≤1000-fold) of reduced function toward physiologic substrates and inhibitors. The extent of zymogen-like character, including resistance to antithrombin III, correlates well with plasma half-life (<2 minutes to >4 hours). Importantly, biologic function, including that of the most zymogen-like variant (FXa-I16T), was greatly enhanced when bound to FVa membranes. This resulted in improvement of clotting times and thrombin generation in hemophilic plasma. The FXa variants were remarkably effective in mouse injury models. In these systems, the data show that the more active the protease, the more difficult it is to overcome the protective mechanism of circulating inhibitors to achieve a therapeutic benefit. Depending on the treatment situation, the more zymogen-like variants (V17S and I16T) were most useful when given before injury whereas variants exhibiting greater activity but shorter half-lives (I16L and I16M) were most effective when administered after injury. This new class of FXa variants provides a useful and flexible platform for selectively bioengineering biologic function and half-life to target different clinical bleeding scenarios., (© 2015 by The American Society of Hematology.)

Membrane overexpression of ErbB-2/HER2 receptor tyrosine kinase (membrane ErbB-2 (MErbB-2)) has a critical role in breast cancer (BC). We and others have also shown the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. Current anti-ErbB-2 therapies, as with the antibody trastuzumab (Ttzm), target only MErbB-2. Here, we found that blockade of NErbB-2 action abrogates growth of BC cells, sensitive and resistant to Ttzm, in a scenario in which ErbB-2, ErbB-3 and Akt are phosphorylated, and ErbB-2/ErbB-3 dimers are formed. Also, inhibition of NErbB-2 presence suppresses growth of a preclinical BC model resistant to Ttzm. We showed that at the cyclin D1 promoter, ErbB-2 assembles a transcriptional complex with Stat3 (signal transducer and activator of transcription 3) and ErbB-3, another member of the ErbB family, which reveals the first nuclear function of ErbB-2/ErbB-3 dimer. We identified NErbB-2 as the major proliferation driver in Ttzm-resistant BC, and demonstrated that Ttzm inability to disrupt the Stat3/ErbB-2/ErbB-3 complex underlies its failure to inhibit growth. Furthermore, our results in the clinic revealed that nuclear interaction between ErbB-2 and Stat3 correlates with poor overall survival in primary breast tumors. Our findings challenge the paradigm of anti-ErbB-2 drug design and highlight NErbB-2 as a novel target to overcome Ttzm resistance.

The resuscitation-promoting factor (Rpf), a secretory protein first reported in Micrococcus luteus, plays a critical role in mycobacterial survival and infection. There are five functionally redundant Rpf-like proteins identified in M. tuberculosis (Mtb). All these Rpfs share a conserved Rpf domain (Rpfd) composed of approximately 70 amino acids, which possesses the same biological functions as the full-length Rpf protein. Glutamic acid at position 54 in Rpfd (E54) has been implicated in mediating multiple physiological processes, and a single amino acid substitution at residue E54 can affect the protein biological activity. In order to determine the effects of different amino acid substitutions of E54 in Rpfd on its immunogenic activity, we generated three recombinant Rpfd mutants, Rpfd1 (E54K), Rpfd2 (E54A) and Rpfd3 (E54K and D48A), based on T-cell epitope prediction and tested their potential protective/therapeutic effects against Mtb in mice. Our results demonstrated that replacement of E54 by different amino acids in Rpfd distinctively influenced its resuscitation-promoting activities and Th1-type immune responses induced in mice. Administration of Rpfd2 mutant enhanced Th1-type cellular responses (IFN-γ and IL-2) in mice (P < 0.05, Rpfd2 versus control) and provided effective protection against Mtb in mice by significantly inhibiting the growth of Mtb during the initial stage of infection. Four weeks after the challenge, the slightest pathological injury in lung was observed in the Rpfd2-immunized group among all three Rfpd mutant-immunized groups. Furthermore, Rpfd2 therapy significantly decreased the bacterial load in lung and alleviated histopathological damage in Mtb-infected mice. Together, our results suggest Rpfd2 as a novel effective vaccine candidate against Mtb., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Emerging successful clinical data on gene therapy using adeno-associated viral (AAV) vector for hemophilia B (HB) showed that the risk of cellular immune response to vector capsid is clearly dose dependent. To decrease the vector dose, we explored AAV-8 (1-3 × 10(12) vg/kg) encoding a hyperfunctional factor IX (FIX-Padua, arginine 338 to leucine) in FIX inhibitor-prone HB dogs. Two naïve HB dogs showed sustained expression of FIX-Padua with an 8- to 12-fold increased specific activity reaching 25% to 40% activity without antibody formation to FIX. A third dog with preexisting FIX inhibitors exhibited a transient anamnestic response (5 Bethesda units) at 2 weeks after vector delivery following by spontaneous eradication of the antibody to FIX by day 70. In this dog, sustained FIX expression reached ∼200% and 30% of activity and antigen levels, respectively. Immune tolerance was confirmed in all dogs after challenges with plasma-derived FIX concentrate. Shortening of the clotting times and lack of bleeding episodes support the phenotypic correction of the severe phenotype, with no clinical or laboratory evidence of risk of thrombosis. Provocative studies in mice showed that FIX-Padua exhibits similar immunogenicity and thrombogenicity compared with FIX wild type. Collectively, these data support the potential translation of gene-based strategies using FIX-Padua for HB., (© 2015 by The American Society of Hematology.)

Random mutagenesis of mouse leptin antagonist (L39A/D40A/F41) followed by selection of high-affinity mutants by yeastsurface display indicated that replacing residue D23 with a non-negatively charged amino acid (most specifically with Leu) leads to dramatically enhanced affinity of leptin toward LEPR leading to development of superactive mouse, human, ovine and rat leptin antagonists (D23L/L39A/D40A/F41A). Superactive leptin antagonist mutants of mouse, human, rat or ovine leptins were developed in our laboratory, expressed in E. coli, refolded and purified to homogeneity as monomeric proteins. Pegylation of leptin antagonists resulted in potent and effective long-acting reagents suitable for in vivo studies or therapies. In the present review we explain the mechanism of leptin inhibition and summarize the possible use of leptin antagonists as possible leptin blockers in various human pathologies such as antiinflammatory and anti-autoimmune diseases, uremic cachexia, and cancer. We also suggest the use of leptin antagonists as research reagents for creation of a novel, fast and reversible model of T2DM in mice.

Using gain-of-function factor IX (FIX) for replacement therapy for haemophilia B (HB) is an attractive strategy. We previously reported a high-activity FIX, FIX-Triple (FIX-V86A/E277A/R338A) as a good substitute for FIX-WT (wild-type) in protein replacement therapy, gene therapy, and cell therapy. Here we generated a new recombinant FIX-TripleL (FIX-V86A/E277A/R338L) by replacing the alanine at residue 338 of FIX-Triple with leucine as in FIX-Padua (FIX-R338L). Purified FIX-TripleL exhibited 22-fold higher specific clotting activity and 15-fold increased binding affinity to activated FVIII compared to FIX-WT. FIX-TripleL increased the therapeutic potential of FIX-Triple by nearly 100% as demonstrated with calibrated automated thrombogram and thromboelastography. FIX-TripleL demonstrated a normal clearance rate in HB mice. The clotting activity of FIX-TripleL was consistently 2- to 3-fold higher in these mice than that of FIX-Triple or FIX-R338L. Gene delivery of adeno-associated virus (AAV) in HB mice showed that FIX-TripleL had 15-fold higher specific clotting activity than FIX-WT, and this activity was significantly better than FIX-Triple (10-fold) or FIX-R338L (6-fold). At a lower viral dose, FIX-TripleL improved FIX activity from sub-therapeutic to therapeutic levels. Under physiological conditions, no signs of adverse thrombotic events were observed in long-term AAV-FIX-treated C57Bl/6 mice. Hepatocellular adenomas were observed in the high- but not the medium- or the low-dose AAV-treated mice expressing FIX-WT or FIX-Triple, indicating the advantages of using hyperfunctional FIX variants to reduce viral doses while maintaining therapeutic clotting activity. Thus, incorporation of the FIX Padua mutation significantly improves the clotting function of FIX-Triple so as to optimise protein replacement therapy and gene therapy.

Objectives: The aim of this study was to characterize the conformation and potency of a mutated glucagon-like peptide-1 (mGLP-1), and evaluate its glucose-lowering activity in diabetic mice., Methods: Spectroscopy techniques were employed to characterize the conformation of mGLP-1. Glucose tolerance test was performed to determine the potency of mGLP-1 in vivo. A mouse model in which diabetes was induced by multiple low doses of streptozotocin was established to evaluate the glucose-lowering activity of mGLP-1., Key Findings: Compared with native GLP-1, mGLP-1 had a similar conformation and an enhanced potency in vivo. In diabetic mice, mGLP-1 displayed a significantly improved glucose-lowering activity as judged by fasting glucose and insulin, oral glucose tolerance test, beta cell function analysis and histochemical analysis., Conclusions: Collectively, mGLP-1 possesses an improved glucose-lowering activity in vivo and therefore can be recognized as a potential candidate for the future development of anti-diabetic drugs., (© 2012 The Authors. JPP © 2012. Royal Pharmaceutical Society.)

Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell-mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo.

The cytokine lymphotoxin-α (LTα) is a promising candidate for use in cancer therapy. However, the instability of LTαin vivo and the insufficient levels of tumor necrosis factor receptor 1 (TNFR1)-mediated bioactivity of LTα limit its therapeutic potential. Here, we created LTα mutants with increased TNFR1-mediated bioactivity by using a phage display technique. We constructed a phage library displaying lysine-deficient structural variants of LTα with randomized amino acid residues. After affinity panning, we screened three clones of lysine-deficient LTα mutant, and identified a LTα mutant with TNFR1-mediated bioactivity that was 32 times that of the wild-type LTα (wtLTα). When compared with wtLTα, the selected clone showed augmented affinity to TNFR1 due to slow dissociation rather than rapid association. In contrast, the mutant showed only 4 times the TNFR2-mediated activity of wtLTα. In addition, the LTα mutant strongly and rapidly activated caspases that induce TNFR1-mediated cell death, whereas the mutant and wtLTα activated nuclear factor-kappa B to a similar extent. Our data suggest that the kinetics of LTα binding to TNFR1 play an important role in signal transduction patterns, and a TNFR1-selective LTα mutant with augmented bioactivity would be a superior candidate for cancer therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Immunotherapy has been used to improve patient immune function, inhibit tumor growth and has become a highly promising method of cancer treatment. Highly agglutinative staphylococcin (HAS), a mixture of Staphylococcus aureus culture filtrates, which include staphylococcal enterotoxin (SE) C as the active ingredient, has been used clinically as an immunomodifier in the treatment of a number of tumors for many years. However, the use of HAS has been associated with some unavoidable side-effects such as fever. Previous studies have shown that SEB stimulates a more potent activation of T lymphocytes than SEC3, and mutations of the histidine residues eliminated the toxicity of SEB. SE mutants with decreased side-effects and/or more potent antitumor activities are required., Methodology/principal Findings: We built a structural model of the MHC II-SEB-TCR complex and found that a mutation of SEB at Lys173 might decrease the repulsion force between the SEB-TCR, which would facilitate their interaction. From the above results, we designed SEB-H32Q/K173E (mSEB). Analysis of in vitro stimulation of the proliferation of human peripheral blood mononuclear cells (PBMCs), IFN-γ secretion and inhibition of the growth of various tumor cell lines demonstrated that mSEB exhibited higher antitumor activity compared with wild-type SEB (wtSEB). Notably, mSEB inhibited the growth of various tumors at an extremely low concentration with little cytotoxicity against normal cells. Three animal tumor models (C57BL/6 mouse, New Zealand rabbit and a humanized NOD/SCID mouse) were used to evaluate the in vivo immunotherapeutic effects. Compared with wtSEB, mSEB significantly enhanced antitumor effect in more than one animal model with reduced pyrexia toxicity and prolonged the survival of tumor-bearing mice., Conclusions/significance: Our results suggest that SEB-H32Q/K173E retains superantigen (SAg) characteristics and enhances the host immune response to neoplastic diseases while reducing associated pyrogenic toxicity.

Objective: Deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) mutants have been reported to exert suicide gene effects in combined gene/chemotherapy of cancer. Here, we aimed to further evaluate the capacity of the mutanted enzyme and its potential for inhibiting cancer cell growth., Methods: We altered the sequence of the last 10 amino acids of Dm-dNK to perform site-directed mutagenesis and constructed active site mutanted Dm-dNK (Dm-dNKmut), RT-PCR and western bloting studies were used to reveal the expression of lentivirus mediated Dm-dNKmut in a breast cancer cell line (Bcap37), a gastric cancer cell line (SGC7901) and a colorectal cancer cell line (CCL187). [3H]-labeled substrates were used for enzyme activity assays, cell cytotoxicity was assessed by MTT assays, cell proliferation using a hemocytometer and apoptosis induction by thenannexin-V-FITC labeled FACS method. In vivo, an animal study was set out in which BALB/C nude mice bearing tumors were treated with lentivirus mediated expression of Dm-dNKmut with the pyrimidine nucleoside analog brivudine (BVDU, (E)-5-(2-bromovinyl)-(2-deoxyuridine)., Results: The Dm-dNKmut could be stably expressed in the cancer cell lines and retained its enzymatic activity. Moreover, the cells expressing Dm-dNKmut exhibited increased sensitivity in combination with BVDU, with induction of apoptosis in vitro and in vivo., Conclusion: These findings underlined the importance of BVDU phosphorylated by Dm-dNKmut in transduced cancer cells and the potential role of Dm-dNKmut as a suicide gene, thus providing the basis for future intensive research for cancer therapy.

The fibroblast growth factor (FGF) signaling pathway is a recognized target of cancer therapy. We have developed a strong inhibitor (S252W mutant soluble ectodomain of FGF recptor-2 IIIc, msFGFR2) that binds FGFs and blocks the activation of FGFRs. Thermodynamic binding studies indicated that msFGFR2 bound FGF-2 16.9 times as strongly as wild-type soluble FGFR2IIIc ectodomain (wsFGFR2). It successfully suppressed the growth, angiogenesis, and metastasis of two tumor cell lines in vitro and in vivo, and it potently inhibited cancer cell proliferation but not normal cell proliferation. Therefore, msFGFR2 is a useful probe for FGF-dependent signaling pathways and a potential broad-spectrum antitumor agent.

Objective: To study the antitumor effects and mechanism of cationic liposome-mediated SurvivinT34A mutants in mice with pulmonary metastases of melanoma B16., Methods: The pulmonary metastases model of B16, a mouse melanoma cell line, was established by the injection of B16 cell suspension via tail vein of C57BL/6 mouse. 3 days later, 18 tumor-bearing mice were randomly divided into 3 groups, which were intravenously administrated with normal saline (100 microL), vector (5 microg DNA, 100 microL) and recombinant plasmid SurvivinT34A mutants (5 microg DNA, 100 microL) every other day for five doses, respectively. After 28 days, the mice were sacrificed, the lung tissue was weighted, and the number of metastasis foci on lung surface was counted and measured. Then, pathological change of lung tissue was studied with HE stainning. The number of apoptotic cells in tumor tissues was assessed by TUNEL assay., Results: Compared with the control, mice treated with mSurvivnT34A had an intact structure of lung, with significant reduction in the number of metastasis foci on lung surface (P < 0.05), and high level of apoptosis in tumor tissue (P < 0. 05)., Conclusion: Recombinant plasmid SurvivinT34A mutants (pORF9-mSurvivinT34A) may inhibit the formation of pulmonary metastases of melanoma.

Multiple sclerosis (MS) is an inflammatory demyelinating disease, the pathogenesis of which is related to elevated serum levels of tumor necrosis factor-α (TNF). Although anti-TNF therapy has been tested as a potential treatment for MS, no remission of symptoms was observed. Recent reports indicated that the TNFR1 signal was responsible for the pathogenesis of murine experimental autoimmune encephalomyelitis (EAE), while the TNFR2 signal was responsible for recovery of the pathogenesis of EAE. Therefore, selective blocking of TNFR1 appears to be a promising strategy for the treatment of MS. In this regard, we previously succeeded in developing a novel TNFR1-selective antagonistic TNF mutant (R1antTNF) by using phage display technology. Here, we have examined the therapeutic potential of R1antTNF using EAE mice. Treatment with PEGylated R1antTNF (PEG-R1antTNF) significantly improved the clinical score and cerebral demyelination at the onset of EAE. Considerable suppression of Th1 and Th17-type response was also observed in spleen and lymph node cells of mice given PEG-R1antTNF. Moreover, the administration of PEG-R1antTNF suppressed the infiltration of inflammatory cells containing Th1 and Th17 cells into the spinal cord. These results suggest that selective blocking of TNFR1 by PEG-R1antTNF could be an effective therapeutic strategy against MS., (Copyright © 2009 Elsevier B.V. All rights reserved.)

Viral myocarditis, which is most prevalently caused by Coxsackievirus B3 (CVB3) infection, affects about 5-20% of the world population and lacks efficient treatments. We previously reported that monocyte chemotactic protein-1 (MCP-1, CCL2) was significantly induced during CVB3 infection and greatly contributed to the myocardic inflammation and injury. Herein a CCL2 mutant with removed chemotactic activity was administrated and its therapeutic effect on CVB3-induced myocarditis was explored. A dominant negative CCL2 mutant, lacking the N-terminal amino acids 2-8 (CCL2(Δ2-8)), was genetically constructed and intramuscularly injected into BALB/c mice after CVB3 infection, severity of myocarditis was evaluated by weight loss, survival rate, serological indices and pathological observation. Systemic and local Th1/Th2 cytokine profiles were also assessed. Mice receiving pCCL2(Δ2-8) exhibited a profound attenuation of myocarditis compared to pcDNA3.1 or non-treated mice, as evidenced by invariant body weight, decreased serum CK-MB level, reduced myocardial inflammatory infiltration and increased survival. This effect was not attributable to the efficient viral clearance, but associated with weakened Th1 immune responses, as evidenced by significantly reduced CD4(+)IFN-γ(+) T cell frequency and Th1 cytokine level systemically and locally. Strategy of blocking in vivo CCL2 activity could effectively alleviate the severity of CVB3-induced myocarditis and may present an alternative therapeutic approach against viral myocarditis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Neuregulin-1 (NRG1), now in a phase II clinical trial, has beneficial effects on heart failure patients through the activation of ErbB2/ErbB4 receptor pair. To decrease the side effect of NRG1 on activating ErbB3, a mutation screen was carried out to get NRG1 mutants, which have more specific binding to ErbB2/ErbB4 receptor pair. Two CHO stable cell lines were constructed, which express ErbB2/ErbB3 or ErbB2/ErbB4 receptor pair, respectively. The ErbB2/ErbB4 cell line showed similar characteristics in ligand-binding activity and the activation of downstream signaling molecules, such as the AKT and PI3K to the primary neonatal rat ventricular myocytes (NRVM), which endogenously expresses ErbB2/ErbB4. Both cell lines have good dose-response. Thirty-one NRG1 mutants were successfully expressed in Escherichia coli and purified. Their ability to stimulate the downstream signaling was measured by detecting AKT phosphorylation. Some mutants showed more specific activation activity in ErbB2/ErbB4 cells. Further study on five of these mutants demonstrated that the change of the activation activity is associated with that of their binding activities to ErbB2/ErbB4 and ErbB2/ErbB3. Four of the candidates are more specific ligands for ErbB2/ErbB4 activation, and thus may serve as more potent drug candidates for heart failure.

Bone morphogenetic proteins (BMPs) are growth factors belonging to the TGF beta super family. To date, more than twenty human BMPs have been identified. Of these, BMP-2 and BMP-7 (also known as osteogenic protein 1 or OP-1) are the only BMPs used clinically. Recombinant forms of both proteins are currently being implanted surgically to induce spinal fusion and to treat long bone non-union fractures. However, in both indications, large quantities of recombinant proteins are needed to induce new bone formation. This translates to higher costs and potential safety risks. Various genetic engineering approaches are being considered to produce second generation BMPs with improved safety and efficacy profiles. Modified BMPs with one or more of the following characteristics are being considered: (i) improved binding affinity to specific target cell surface BMP receptors, (ii) decreased sensitivity to natural BMP inhibitors, (iii) better immunogenicity profile, and (iv) increased solubility and stability, to cite a few. This review summarizes the progress made so far in this field and gives a perspective on what the next generation BMPs could look like.

Lung cancer is a leading cause of cancer death due to the high incidence of metastasis; therefore, novel and effective treatments are urgently needed. A current strategy is cancer-specific targeted gene therapy. Although many identified that cancer-specific promoters are highly specific, they tend to have low activity compared with the ubiquitous cytomegalovirus (CMV) promoter, limiting their application. We developed a targeted gene therapy expression system for lung cancer that is highly specific with strong activity. Our expression vector uses the survivin promoter, highly expressed in many cancers but not normal adult tissues. We enhanced the survivin promoter activity comparable to the CMV promoter in lung cancer cell lines using an established platform technology, whereas the survivin promoter remained weak in normal cells. In mouse models, the transgene was specifically expressed in the lung tumor tissue, compared with the CMV promoter that was expressed in both normal and tumor tissues. In addition, the therapeutic gene BikDD, a mutant form of pro-apoptotic Bcl2 interacting killer, induced cell killing in vitro, and inhibited cell growth and prolonged mouse survival in vivo. Importantly, there was virtually no toxicity when BikDD was expressed with our expression system. Thus, the current report provides a therapeutic efficacy and safe strategy worthy of development in clinical trials treating lung cancer.

We used a xenograft model to investigate whether the aryl hydrocarbon receptor deletion construct CDelta553 suppresses tumor growth. HeLa cells that were infected with CDelta553 expressing adenovirus (Ad553) formed very small tumors whereas the control adenovirus-infected cells formed large tumors at day 15. CDelta553 inhibited the formation of the HIF-1 DNA complex and suppressed the induction of the HIF-1alpha target proteins CAIX and GLUT1. The Ad553 tumors had less HIF-1 function since they showed reduced microvessel formation and lesser amounts of HIF-1alpha, Arnt, phospho-Akt, CAIX, and GLUT1. Proteasome-mediated Arnt degradation was enhanced in Ad553-infected HeLa cells and tumors.

Cytosine deaminase is used in combination with 5-fluorocytosine as an enzyme-prodrug combination for targeted genetic cancer treatment. This approach is limited by inefficient gene delivery and poor prodrug conversion activities. Previously, we reported individual point mutations within the substrate binding pocket of bacterial cytosine deaminase (bCD) that result in marginal improvements in the ability to sensitize cells to 5-fluorocytosine (5FC). Here, we describe an expanded random mutagenesis and selection experiment that yielded enzyme variants, which provide significant improvement in prodrug sensitization. Three of these mutants were evaluated using enzyme kinetic analyses and then assayed in three cancer cell lines for 5FC sensitization, bystander effects, and formation of 5-fluorouracil metabolites. All variants displayed 18- to 19-fold shifts in substrate preference toward 5FC, a significant reduction in IC(50) values and improved bystander effect compared with wild-type bCD. In a xenograft tumor model, the best enzyme mutant was shown to prevent tumor growth at much lower doses of 5FC than is observed when tumor cells express wild-type bCD. Crystallographic analyses of this construct show the basis for improved activity toward 5FC, and also how two different mutagenesis strategies yield closely related but mutually exclusive mutations that each result in a significant alteration of enzyme specificity.

The MCT-1 oncogene, highly expressed in a subset of non-Hodgkin's lymphomas interacts with the cap complex through its PUA domain. MCT-1 recruits DENR, a SUI1 motif containing protein that promotes translation initiation of cancer-related mRNAs. We reasoned that a PUA-domain mutant protein would repress MCT-1 function and attenuate the malignant phenotype. Human lymphoma cell lines expressing the PUA-domain mutant protein demonstrated reduced anchorage-independent growth and increased susceptibility to apoptosis. Significantly, we identified an altered translational profile in cells expressing the mutant protein. These data further buttress the role of the MCT-1 in lymphomagenesis and support the development of novel therapeutic strategies targeting MCT-1.

The combination of molecular chemotherapy with radiation therapy has the potential to become a powerful approach for treatment of pancreatic cancer. We have developed an adenoviral vector (AdbCD-D314A) encoding a mutant bacterial cytosine deaminase (bCD) gene, which converts the prodrug 5-fluorocytosine (5-FC) into the active drug 5-fluorouracil. The aim of this study was to investigate AdbCD-D314A/5-FC-mediated cytotoxicity in vitro and therapeutic efficacy in vivo alone and in combination with radiation against human pancreatic cancer cells and xenografts. AdbCD-D314A/5-FC-mediated cytotoxicity alone and in combination with radiation was analyzed using crystal violet inclusion and clonogenic survival assays. CD enzyme activity was determined by measuring conversion of [3H]5-FC to [3H]5-fluorouracil after adenoviral infection of pancreatic cancer cells in vitro and pancreatic tumor xenografts by TLC. S.c. pancreatic tumor xenografts were used to evaluate the therapeutic efficacy of AdbCD-D314A/5-FC molecular chemotherapy in combination with radiation therapy. AdbCD-D314A infection resulted in increased 5-FC-mediated pancreatic cancer cell killing that correlated with significantly enhanced CD enzyme activity compared with AdbCDwt encoding wild-type of bCD. Animal studies showed significant inhibition of growth of human pancreatic tumors treated with AdbCD-D314A/5-FC in comparison with AdbCDwt/5-FC. Also, a significantly greater inhibition of growth of Panc2.03 and MIA PaCA-2 tumor xenografts was produced by the combination of AdbCD-D314A/5-FC with radiation compared with either agent alone. The results indicate that the combination of AdbCD-D314A/5-FC molecular chemotherapy with radiation therapy significantly enhanced cytotoxicity of pancreatic cancer cells in vitro and increased therapeutic efficacy against human pancreatic tumor xenografts.

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), a cytokine belonging to the IL-10 family, displays cancer-specific apoptosis-inducing properties when delivered by a replication-incompetent adenovirus (Ad.mda-7) or as a GST-tagged recombinant protein (GST-MDA-7). Previous studies demonstrated that an adenovirus expressing M4, a truncated version of MDA-7/IL-24 containing amino acid residues 104-206, also induced similar cancer-specific apoptosis. We generated recombinant GST-M4 proteins and examined the potency of GST-MDA-7 and GST-M4 on a panel of epidermal growth factor receptor (EGFR) wild type and mutant non-small cell lung carcinoma (NSCLC) cells either as a single agent or in combination with a reversible EGFR inhibitor, Tarceva. The combination of either GST-MDA-7 or GST-M4 ( approximately 0.1 microM) and Tarceva (10 microM), at sub-optimal apoptosis-inducing concentrations synergistically enhanced growth inhibition and apoptosis induction over that observed with either agent alone. The combination treatment also augmented inhibition of EGFR signaling, analyzed by phosphorylation of EGFR and its downstream effectors AKT and ERK1/2, over that with single-agent therapy. Tarceva enhanced GST-MDA-7 and GST-M4 toxicity in cells expressing mutated EGFR proteins that are resistant to the inhibitory effects of Tarceva. In total, these data suggest that combined treatment of NSCLC cells with an EGFR inhibitor can augment the efficacy of GST-MDA-7 and GST-M4 and that the EGFR inhibitor Tarceva may mediate this combinatorial effect by inhibiting multiple tyrosine kinases in addition to the EGFR. This approach highlights a potential new combinatorial strategy, which may prove beneficial for NSCLC patients with acquired resistance to EGFR inhibitors., ((c) 2008 Wiley-Liss, Inc.)