Seong-Hyun Ho

Electro-gene therapy of collagen-induced arthritis by using an expression plasmid for the soluble p75 tumor necrosis factor receptor-Fc fusion protein.

Tumor necrosis factor (TNF) is a proinflammatory cytokine involved in the pathogenesis of rheumatoid arthritis, and antagonism of TNF may reduce the activity of the disease. Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. In this study, we attempted to treat collagen-induced arthritis (CIA) with anti-TNF gene therapy by transferring the plasmid encoding soluble p75 TNF receptor linked to the Fc portion of human IgG1 (sTNFR:Fc) using in vivo electroporation. DBA/1 mice were immunized with bovine type II collagen and boosted with the same antigen. At 2 days after boosting, the plasmid vector containing cDNA for the sTNFR:Fc was injected into one selected site in the gastrocnemius muscle followed by electroporation. Serum levels of sTNFR:Fc reached 2.3 ng/ml on day 5 when gene expression reached its peak. Macroscopic analysis of paws for redness, swelling and deformities showed that the onset of moderate-to-severe CIA in mice treated with sTNFR:Fc was prevented on a significant level compared with the control mice (P<0.05). The beneficial effect of sTNFR:Fc DNA transfer lasted for at least 18 days following treatment. In addition, both the synovitis and the erosion of cartilage in the knee joints were dramatically reduced in mice treated with sTNFR:Fc (P<0.05). The expression of IL-1β and IL-12 in the paw was also decreased by sTNFR:Fc treatment (P<0.01) while there was little change in the levels of IL-17 and vWF. These data showed that sTNFR:Fc expression plasmid was effective in the prevention of CIA, and in vivo electroporation-mediated gene transfer may provide a new approach to cytokine therapy in autoimmune arthritis.

Protection against collagen-induced arthritis by intramuscular gene therapy with an expression plasmid for the interleukin-1 receptor antagonist

The interleukin-1 receptor antagonist (IL-1Ra) is an endogenous protein that can prevent the binding of IL-1 to its cell-surface receptors. Among a number of techniques for gene transfer in vivo, the direct injection of naked DNA into muscle is simple, inexpensive and safe. In this study, we evaluated the potential of intramuscular gene therapy with plasmid DNA containing the cDNA for IL-1Ra in the prevention of murine collagen-induced arthritis (CIA). DBA/1 mice were immunized with bovine type II collagen. At 4 weeks after the initial immunization, expression plasmid for IL-1Ra was injected into four selected sites in the thigh and calf muscles of DBA/1 mice. Control mice received the same plasmid, but lacking the IL-1Ra coding sequence. Macroscopic analysis of paws for redness, swelling and deformities showed that the onset of moderate to severe CIA in the paws of mice injected with IL-1Ra DNA was significantly prevented (P<0.05). In addition, both the synovitis and the cartilage erosion in knee joints were dramatically reduced in mice treated with IL-1Ra DNA (P<0.05). The expression of IL-1β was significantly decreased in the ankle joints of mice treated with IL-1Ra (P<0.01). Interestingly, the levels of IL-1Ra in sera and joints after intramuscular injection of IL-1Ra DNA were significantly lower than when protein had been used in previous reports, suggesting that the therapeutic effect may be achieved by an alternative mechanism(s) rather than by systemic elevation of IL-1Ra. These observations provide the first evidence that direct intramuscular injection of expression plasmid for IL-1Ra may effectively suppress the inflammatory pathology in arthritis.

A phase I clinical study of naked DNA expressing two isoforms of hepatocyte growth factor to treat patients with critical limb ischemia.

The purpose of the present phase I clinical trial was to evaluate the safety, tolerability, and preliminary efficacy of naked DNA therapy expressing two isoforms of hepatocyte growth factor (pCK‐HGF‐X7) in critical limb ischemia (CLI) patients.

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL‐1Ra for the prevention of murine collagen‐induced arthritis (CIA).

Viral vector-mediated transduction of a modified thrombospondin-2 cDNA inhibits tumor growth and angiogenesis.

Gene therapy represents a possible alternative to the chronic delivery of recombinant antiangiogenic proteins to cancer patients. We have constructed retroviral and adenoviral vectors that express murine N-terminal fragments of thrombospondin-2 (NfTSP2), a potent endogenous inhibitor of tumor growth and angiogenesis. To test the possibility of anticancer gene therapy using NfTSP2, we tested whether an ex vivo retrovirus-mediated procedure could be used for the treatment of tumors. The treatment of tumor-bearing mice with syngenic immortalized cell lines expressing NfTSP2 led to a tumor volume reduction up to 70% as compared with the controls (P<0.005). In addition, the established tumors were eradicated in 40% of the mice treated with NfTSP2-expressing cells. Furthermore, the intratumoral injection of the NfTSP2-expressing adenoviral vector to the human squamous cell carcinoma in nude mice resulted in a significant reduction of the growth rates and the volumes of the carcinoma (P<0.05). Immunohistochemical staining of the tumors indicated that the total area and the average size of tumor vessels were significantly reduced in the treatment group versus the controls (P<0.05). In conclusion, the present study clearly demonstrates that the viral vector-mediated transfer of the NfTSP2 gene could inhibit the growth of tumors by perturbing tumor-associated angiogenesis.

Improvement of biological and pharmacokinetic features of human interleukin-11 by site-directed mutagenesis.

Recombinant human interleukin-11 (rhIL-11) has been shown to increase platelet counts in animals and humans and is the only drug approved for its use in chemotherapy-induced thrombocytopenia (CIT). However, due to its serious side effects, its clinical use has been limited. The current work presents significantly improved efficacy of rhIL-11 via knowledge based re-designing process. The interleukin-11 mutein (mIL-11) was found to endure chemical and proteolytic stresses, while retaining the biological activity of rhIL-11. The improved efficacy of mIL-11 was evident after subcutaneous administration of mIL-11 and rhIL-11 in the rodent and primate models. More than three-fold increase in maximum plasma concentration (Cmax) and area-under-the curve (AUC) was observed. Furthermore, three-fold higher increase in the platelet counts was obtained after seven consecutive daily subcutaneous mIL-11 injections than that with rhIL-11. The mIL-11 demonstrated not only improved stability but also enhanced efficacy over the currently used rhIL-11 regimen, thereby suggesting less toxicity.

Effect of HX108-CS supplementation on exercise capacity and lactate accumulation after high-intensity exercise.

BackgroundIn the present study, we determined the effects of HX108-CS (mixed extract of Schisandra chinensis and Chaenomeles sinensis) supplementation on lactate accumulation and endurance capacity. Furthermore, we examined CK (creatine kinase), LDH (lactate dehydrogenase) activity to determine whether the HX108-CS affected markers of skeletal muscle injury in vivo and in vitro.MethodsExercise capacity was measured by an exhaustive swimming test using ICR mice divided into four groups; one group received distilled water (DW) (Control group, n = 10), and the other groups received three different dosages of HX108-CS (10, 50 and 100 mg/kg, n = 10 per group) solution in water orally. Then, for the time-dependent measurements of blood lactate, CK, and LDH, Sprague–Dawley rats were divided into two groups; one received DW (Control group, n = 10), and the other group received HX108-CS (100 mg/kg, n = 10) solution in the same way as mice. Before the exercise test, the animals were given either DW or HX108-CS for 2 weeks. High-intensity treadmill exercise was performed for 30 minutes. Blood samples were collected and analyzed during and after exercise. For the in vitro experiment, C2C12 cells were treated with HX108-CS to examine its effect on lactate production, CK, and LDH activity.ResultsBlood lactate concentration was significantly lowered immediately after treadmill exercise in HX108-CS group; however, there were no significant differences in activities of CK and LDH between HX108-CS and control during treadmill exercise and recovery phase. Furthermore, treatment with 100 mg/kg of HX108-CS led to a significant increase in the time to exhaustion in swimming test, and concurrently blood lactate concentration was significantly decreased in 50 and 100 mg/kg treated group. Moreover, our results of in vitro experiment showed that HX108-CS suppressed lactate production, CK, and LDH activity in a dose-dependent manner.ConclusionsThese results suggest that supplementation with HX108-CS may enhance exercise capacity by lowering lactate accumulation. This may in part be related to an amelioration of skeletal muscle injury.

Immunogenicity and safety profiles of genetic vaccines against human Her-2/neu in cynomolgus monkeys.

Her-2/neu is a well-characterized tumor-associated antigen, the overexpression of which in human carcinomas correlates with a poor prognosis. Here, we evaluated Her-2/neu-specific humoral and cellular immune responses in immunized monkeys after immunization with nonreplicating adenovirus (AdHM) expressing the extracellular and transmembrane domain of human Her-2/neu (HM) and/or naked DNA vaccine (pHM-hGM-CSF) expressing human granulocyte-macrophage colony-stimulating factor together with HM. Priming of monkeys with AdHM generated Her-2/neu-specific long-lasting antibody production. Furthermore, these Her-2/neu-specific antibodies produced by AdHM immunization, some of which shared epitope specificity with Herceptin, were able to induce antibody-dependent cellular cytotoxicity against Her-2-expressing target cells. Cellular immune responses were elicited in all monkeys immunized with Her-2/neu-expressing vaccine; interferon-γ was secreted when these splenocytes were restimulated with Her-2/neu-expressing autologous cells, and immunization with AdHM induced Her-2/neu-specific lymphoproliferative responses. Further, immunization with pHM-hGM-CSF before AdHM immunization noticeably enhanced cytotoxic T-lymphocyte activity. In addition, we observed no abnormalities that would indicate that the genetic vaccines had toxic effects in the immunized monkeys. Thus, we can conclude that our genetic vaccines efficiently elicited Her-2/neu-specific humoral and cellular immune responses without causing severe adverse effects in nonhuman primates and that as such they warrant further clinical investigation.

The pharmacokinetic and pharmacodynamic properties of site-specific pegylated genetically modified recombinant human interleukin-11 in normal and thrombocytopenic monkeys

Graphical abstract Figure. No Caption available. Abstract In order to improve the pharmacokinetic and pharmacodynamic properties of recombinant human interleukin‐11 mutein (mIL‐11) and to reduce the frequency of administration, we examined the feasibility of chemical modification of mIL‐11 by methoxy polyethylene glycol succinimidyl carbonate (mPEG‐SC). PEG‐mIL‐11 was prepared by a pH controlled amine specific method. Bioactivity of the protein was determined in a IL‐11‐dependent in vitro bioassay, its pharmacodynamic and pharmacokinetic properties were investigated by using normal and thrombocytopenic monkey models. N‐terminus sequencing and peptide mapping analysis revealed that Lys33 is the PEGylated position for PEG‐mIL‐11. Bioactivity of PEG‐mIL‐11 assessed by B9‐11 cell proliferation assay was comparable to that of mIL‐11. More than 79‐fold increase in area‐under‐the curve (AUC) and 26‐fold increase in maximum plasma concentration (Cmax) was observed in pharmacokinetic analysis. Single dose administration of the PEG‐mIL‐11 induced blood platelets number increase and the effect duration were comparable to that of 7 to 10 consecutive daily administration of mIL‐11 to the normal and thrombocytopenic monkey models. PEG‐mIL‐11 is a promising therapeutic for thrombocytopenia.

A phase II clinical study of interleukin-11 mutein to treat patients with chemotherapy-induced thrombocytopenia.

9113 Background: To demonstrate the safety and efficacy of genetically engineered human interleukin-11 mutein (mIL-11) on chemotherapy- induced thrombocytopenia (CIT) patients, a multicenter random...