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In the pursuit of therapeutic strategies for myocardial infarction (MI), a pivotal objective lies in the concurrent restoration of blood perfusion and reduction of cardiomyocyte apoptosis. However, achieving these dual goals simultaneously presents a considerable challenge. In this study, a Zn2SiO4bioceramic capable of concurrently sustaining the release of bioactive SiO32−and Zn2+ions, which exhibit a synergistic impact on endothelial cell angiogenesis promotion, cardiomyocyte apoptosis inhibition, and myocardial mitochondrial protection against oxygen‐free radical (reactive oxygen species) induced injury is developed. Furthermore, in vivo outcomes from a murine MI model demonstrate that either systemic administration via tail vein injection of Zn2SiO4extract or local application through intramyocardial injection of a Zn2SiO4composite hydrogel promotes cardiac function and reduces cardiac fibrosis, thus aiding myocardial repair. This research is the first to elucidate the advantageous effects of dual bioactive ions in myocardial protection and may offer a novel therapeutic avenue for ischemic heart disease based on meticulously engineered bioceramics. The bioactive Zn2+and SiO32−ions derived from Zn2SiO4bioceramic can inhibit pathological cardiac remodeling and promote myocardial repair in mice after myocardial infarction in different forms including ions extract for systemic therapy (tail intravenous injection) and composite hydrogel for local therapy (intramyocardial injection) by promotion of angiogenesis, protection of myocardial mitochondrial, and inhibition of cardiomyocyte apoptosis.

pH-responsive drug delivery systems have attracted great interest because of their potential use in antitumor therapies. Herein, we report a facile one-pot fabrication of a “host−metal−drug” coordination-bonding system in a mesostructured surfactant/silica hybrid for the pH-responsive drug delivery purpose. The mesostructure has been synthesized by self-assembly of nontoxic and biocompatible F127 Pluronic nonionic surfactant and silica source through a real liquid crystal templating route, in which F127 act as host molecules. Metal ions such as Zn, Cu, and Fe and drugs have been introduced simultaneously into the mesostructure synthesis systems, to form F127−metal−drug coordination-bonding architecture. The cleavage of the coordination bonds that are sensitive to variations in external pH gives rise to the release of the drug under weakly acidic conditions (pH 5.0−6.0). To assist in the release of drugs without significant binding capabilities, a vector has been designed to endow coordinately inert drugs with pH-responsive properties. Furthermore, the pH responsibility has been confirmed by cell assay and in vivo tumor test, opening up new opportunities for the potential application as implants in antitumor therapies.

One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells, whereas increased transforming growth factor-β levels in plasma and the frequency of both CD4+CD25+FoxP3+ and CD4+CD25−Foxp3+ regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4+CD25+ cells did not abrogate tolerance induction, adoptive transfer of CD4+ cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3–treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIII plasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-β levels and the generation of adaptive FVIII-specific CD4+Foxp3+ regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+and CD8+T cells, whereas increased transforming growth factor-β levels in plasma and the frequency of both CD4+CD25+FoxP3+and CD4+CD25−Foxp3+regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4+CD25+cells did not abrogate tolerance induction, adoptive transfer of CD4+cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3–treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIIIplasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-β levels and the generation of adaptive FVIII-specific CD4+Foxp3+regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

Gene transfer of a factor VIII (FVIII) plasmid into hemophilia A (HemA) mice achieved supraphysiologic FVIII expression, but triggered production of high-titer FVIII-specific antibodies and loss of functional FVIII activity. To test whether FVIII-specific regulatory T cells (Tregs) can modulate immune responses against FVIII, we developed a HemA mouse model in which all T cells overexpressed Foxp3 (HemA/Foxp3-Tg). FVIII plasmid therapy did not induce antibody production in HemA/Foxp3-Tg mice. CD4+Foxp3+ T cells isolated from plasmid-treated HemA/Foxp3-Tg mice significantly suppressed proliferation of FVIII-stimulated CD4+ effector T cells. The percentage of CD4+ T cells expressing CD25, glucocorticoid-induced tumor necrosis factor receptor, and cytotoxic T lymphocyte antigen 4 increased significantly in spleen and peripheral blood for 9 weeks. Mice receiving adoptively transferred Tregs from FVIII-exposed HemA/Foxp3-Tg mice produced significantly reduced antibody titers compared with controls after initial challenge with FVIII plasmid and second challenge 16 weeks after first plasmid treatment. Adoptively transferred Tregs engrafted and distributed at 2% to 4% in the Treg compartment of blood, lymph nodes, and spleens of the recipient mice and induced activation of endogenous Tregs; the engraftment decreased to negligible levels over 8 to 12 weeks. Antigen-specific Tregs can provide long-lasting protection against immune responses in vivo and limit recall responses induced by a second challenge via infectious tolerance.

Gene transfer of a factor VIII(FVIII) plasmidinto hemophilia A (HemA) mice achieved supraphysiologic FVIII expression, but triggered production of high-titer FVIII-specific antibodies and loss of functional FVIII activity. To test whether FVIII-specific regulatory T cells (Tregs) can modulate immune responses against FVIII, we developed a HemAmouse model in which all T cells overexpressed Foxp3(HemA/Foxp3-Tg). FVIII plasmidtherapy did not induce antibody production in HemA/Foxp3-Tgmice. CD4+Foxp3+T cells isolated from plasmid-treated HemA/Foxp3-Tgmice significantly suppressed proliferation of FVIII-stimulated CD4+effector T cells. The percentage of CD4+T cells expressing CD25, glucocorticoid-induced tumor necrosis factor receptor, and cytotoxic T lymphocyte antigen 4 increased significantly in spleen and peripheral blood for 9 weeks. Mice receiving adoptively transferred Tregs from FVIII-exposed HemA/Foxp3-Tgmice produced significantly reduced antibody titers compared with controls after initial challenge with FVIII plasmidand second challenge 16 weeks after first plasmidtreatment. Adoptively transferred Tregs engrafted and distributed at 2% to 4% in the Treg compartment of blood, lymph nodes, and spleens of the recipient mice and induced activation of endogenous Tregs; the engraftment decreased to negligible levels over 8 to 12 weeks. Antigen-specific Tregs can provide long-lasting protection against immune responses in vivo and limit recall responses induced by a second challenge via infectious tolerance.

Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid–treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS+CD4+ T cells and activation of CD25+Foxp3+ Tregs in the peripheral blood, spleen, and lymph nodes. CD4+ T cells from anti-ICOS–treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-β. Moreover, CD4+CD25+ Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS–treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Φx 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII(hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid–treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS+CD4+T cells and activation of CD25+Foxp3+Tregs in the peripheral blood, spleen, and lymph nodes. CD4+T cells from anti-ICOS–treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-β. Moreover, CD4+CD25+Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIIIplasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS–treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Φx 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

Formation of inhibitory antibodies is a significant problem encountered in the treatment of hemophilia by replacement therapy. Nonviral gene transfer of a factor VIII plasmid into hemophilia mice induced strong humoral responses through predominantly TH2 signals. The plasmid-treated mice produced persistent, high-level inhibitory antibody specifically against FVIII, representing a unique and useful model for testing various immunomodulation strategies. It was previously demonstrated by our group that transient immunosuppression by CTLA4-Ig and anti-CD40L (MR1) can prevent inhibitory antibody formation following nonviral gene transfer of FVIII plasmid into hemophilia A mice. In this study, we tested if blockade of inducible costimulator (ICOS)-ICOS ligand (ICOSL) pathway in combination with or without agents blocking other co-stimulatory pathways can modulate the immune response following gene therapy treatment. Three groups of mice (n=5/group) were subjected to administration of FVIII plasmid via hydrodynamics-based tail-vein injection, and transient immunosuppressive regimens including anti-ICOS (8 treatment in 2 week period), combination of anti-ICOS (same dose) and CTLA4-Ig (2 treatment at day 0 and 2), and combination of anti-ICOS (same dose) and MR1 (5 treatment in 2 week period). 2 mice from anti-ICOS only group, 3 mice from combination treatment of anti-ICOS and CTLA4-Ig group, and 2 mice from combination treatment of anti-ICOS and MR1 group developed inhibitors at 2 weeks post treatment. The rest of the mice did not develop inhibitors. These results imply that neither synergistic nor additional modulation was achieved by combining CTLA4-Ig or MR1 with anti-ICOS compared to anti-ICOS alone. Subsequently a more frequent and longer anti-ICOS treatment (16 treatment in 4 week period) was administered in two separate groups of FVIII plasmid-treated mice (n=5 and 11 per group, respectively). All the treated mice did not produce inhibitory antibodies against FVIII and produced persistent, high-level (100–300 µg/ml) FVIII gene expression for at least 150 days (experimental period). The CD4+ T cells isolated from the spleen of tolerized mice did not proliferate in response to FVIII stimulation in vitro. Furthermore, higher population of CD4+CD25+ regulatory T cells were detected in peripheral blood in the tolerized mice compared to untreated and plasmid-treated mice. Adoptive transfer of CD4+ T cells isolated from tolerized mice is performed to test if these cells can protect the recipient mice from developing inhibitory antibodies against FVIII. Anti-ICOS treatment has the potential for a new immunomodulatory strategy for preventing the formation of inhibitory antibodies against FVIII following gene therapy.

Formation of inhibitory antibodies is a significant problem encountered in the treatment of hemophilia by replacement therapy. Nonviral gene transfer of a factor VIII plasmid into hemophilia mice induced strong humoral responses through predominantly TH2 signals. The plasmid-treated mice produced persistent, high-level inhibitory antibody specifically against FVIII, representing a unique and useful model for testing various immunomodulation strategies. It was previously demonstrated by our group that transient immunosuppression by CTLA4-Ig and anti-CD40L (MR1) can prevent inhibitory antibody formation following nonviral gene transfer of FVIII plasmid into hemophilia A mice. In this study, we tested if blockade of inducible costimulator (ICOS)-ICOS ligand (ICOSL) pathway in combination with or without agents blocking other co-stimulatory pathways can modulate the immune response following gene therapy treatment. Three groups of mice (n=5/group) were subjected to administration of FVIII plasmid via hydrodynamics-based tail-vein injection, and transient immunosuppressive regimens including anti-ICOS (8 treatment in 2 week period), combination of anti-ICOS (same dose) and CTLA4-Ig (2 treatment at day 0 and 2), and combination of anti-ICOS (same dose) and MR1 (5 treatment in 2 week period). 2 mice from anti-ICOS only group, 3 mice from combination treatment of anti-ICOS and CTLA4-Ig group, and 2 mice from combination treatment of anti-ICOS and MR1 group developed inhibitors at 2 weeks post treatment. The rest of the mice did not develop inhibitors. These results imply that neither synergistic nor additional modulation was achieved by combining CTLA4-Ig or MR1 with anti-ICOS compared to anti-ICOS alone. Subsequently a more frequent and longer anti-ICOS treatment (16 treatment in 4 week period) was administered in two separate groups of FVIII plasmid-treated mice (n=5 and 11 per group, respectively). All the treated mice did not produce inhibitory antibodies against FVIII and produced persistent, high-level (100–300 μg/ml) FVIII gene expression for at least 150 days (experimental period). The CD4+ T cells isolated from the spleen of tolerized mice did not proliferate in response to FVIII stimulation in vitro. Furthermore, higher population of CD4+CD25+ regulatory T cells were detected in peripheral blood in the tolerized mice compared to untreated and plasmid-treated mice. Adoptive transfer of CD4+ T cells isolated from tolerized mice is performed to test if these cells can protect the recipient mice from developing inhibitory antibodies against FVIII. Anti-ICOS treatment has the potential for a new immunomodulatory strategy for preventing the formation of inhibitory antibodies against FVIII following gene therapy.