Sang-Oh Han

Water-soluble lipopolymer as an efficient carrier for gene delivery to myocardium

Water-soluble lipopolymer (WSLP), which consisted of polyethylenimine (PEI, 1800 Da) and cholesterol, was characterized as a gene carrier to smooth muscle cells and myocardium. Acid–base titration showed that WSLP had a proton-buffering effect. The size of WSLP/plasmid DNA (pDNA) complex was around 70 nm. WSLP/pDNA complex was transfected to A7R5 cells, a smooth muscle cell line. WSLP showed the highest transfection at a 40/1 N/P ratio. WSLP has higher transfection efficiency than PEI (1800 and 25 000 Da), SuperFect, and lipofectamine. In addition, WSLP has less cytotoxicity than PEI (25 000 Da), SuperFect, and lipofectamine. Since WSLP has cholesterol moiety, it may utilize cellular cholesterol uptake pathway, in which low-density lipoprotein (LDL) is involved. An inhibition study with free cholesterol or low-density lipoprotein (LDL) showed that transfection was inhibited by cholesterol or LDL, suggesting that WSLP/pDNA complex is transfected to the cells through the cholesterol uptake pathway. To evaluate the transfection efficiency to myocardium, WSLP/pDNA complex was injected into the rabbit myocardium. WSLP showed higher transfection than PEI and naked pDNA. WSLP expressed the transgene for more than 2 weeks. In conclusion, WSLP is an efficient carrier for local gene transfection to myocardium, and useful in in vivo gene therapy.

Hypoxia-inducible VEGF gene delivery to ischemic myocardium using water-soluble lipopolymer

Therapeutic angiogenesis with gene encoding vascular endothelial growth factor (VEGF) is a new potential treatment in cardiovascular disease. However, unregulated VEGF-mediated angiogenesis has the potential to promote tumor growth, accelerate diabetic proliferative retinopathy, and promote rupture of atherosclerotic plaque. To be safe and effective, gene therapy with VEGF must be regulated. To limit the risk of pathological angiogenesis, we developed a hypoxia-inducible VEGF gene therapy system using the erythropoietin (Epo) enhancer and water-soluble lipopolymer (WSLP). pEpo-SV-VEGF or pSV-VEGF-Epo was constructed by insertion of the Epo enhancer upstream of the Simian Virus 40 (SV40) promoter or downstream of the poly(A) signal of pSV-VEGF. In vitro transfection showed that pEpo-SV-VEGF, not pSV-VEGF-Epo, induced the VEGF expression in hypoxic cells. In addition, the VEGF protein, which was produced from the Epo-SV-VEGF-transfected and hypoxia-incubated cells, was able to enhance the proliferation of the endothelial cells. Injection of the pEpo-SV-VEGF/WSLP complex showed that the expression of VEGF was induced in ischemic myocardium, compared to normal myo-cardium. Therefore, with the localized induction of VEGF and the low cytotoxicity of WSLP, the pEpo-SV-VEGF/WSLP system may be helpful to eventually treat ischemic heart disease.

Degradable polymeric carrier for the delivery of IL-10 plasmid DNA to prevent autoimmune insulitis of NOD mice

Recently, we have reported that biodegradable poly [α-(4-aminobutyl)-L-glycolic acid] (PAGA) can condense and protect plasmid DNA from DNase I. In this study, we investigated whether the systemic administration of pCAGGS mouse IL-10 (mIL-10) expression plasmid complexed with PAGA can reduce the development of insulitis in non-obese diabetic (NOD) mice. PAGA/mIL-10 plasmid complexes were stable for more than 60 min, but the naked DNA was destroyed within 10 min by DNase I. The PAGA/DNA complexes were injected into the tail vein of 3-week-old NOD mice. Serum mIL-10 level peaked at 5 days after injection, and could be detected for more than 9 weeks. The prevalence of severe insulitis on 12-week-old NOD mice was markedly reduced by the intravenous injection of PAGA/DNA complex (15.7%) compared with that of naked DNA injection (34.5%) and non-treated controls (90.9%). In conclusion, systemic administration of pCAGGS mIL-10 plasmid/PAGA complexes can reduce the severity of insulitis in NOD mice. This study shows that the PAGA/DNA complex has the potential for the prevention of autoimmune diabetes mellitus.

Artery wall binding peptide-poly(ethylene glycol)-grafted-poly(L-lysine)-based gene delivery to artery wall cells.

Artery wall binding peptide (AWBP; Cys-Gly-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Gln-Ala-Glu-Gly-Ala-Lys), a specific targeting peptide, was conjugated to poly(ethylene glycol)-grafted-poly(L-lysine) (PEG-g-PLL) to enhance the gene transfer to artery wall cells. AWBP-PEG-PLL was synthesized by the reaction between the vinylsulfone group of PEG-g-PLL and the thiol group of cysteine in AWBP. 1H-NMR analysis confirmed the composition of the obtained polymer and indicated that four mol of AWBP were reacted to one mole of VS-PEG-PLL. The particles of AWBP-PEG-PLL/pDNA complexes were determined spherical with a size of approximately 100 nm by dynamic light scattering (DLS) and atomic force microscopy (AFM). Agarose gel retardation assay indicated that AWBP-PEG-PLL was able to condense plasmid DNA and reach complete complexation at and above a charge ratio 1/1 (+/-). Transfection efficiency of AWBP-PEG-PLL/pDNA complexes was 150-180 times higher than that of control systems, such as PEG-g-PLL/pDNA and PLL/pDNA, in both bovine aorta endothelial cells and smooth muscle cells. Luciferase activities of AWBP-PEG-PLL depended on the amount of free AWBP, while those of the control carriers such as PLL and PEG-g-PLL were not affected by free AWBP. These results supported that gene transfer of AWBP-PEG-PLL/pDNA complexes to bovine aorta wall cells was mediated by specific artery wall cell receptor-mediated endocytosis.

Biodegradable polymer-based interleukin-12 gene delivery: role of induced cytokines, tumor infiltrating cells and nitric oxide in anti-tumor activity

The objective of this study was to investigate the role of induced cytokines, tumor infiltrating cells and nitric oxide (NO) in anti-tumor activity upon intratumoral injection of free and condensed plasmid DNA encoding murine interleukin-12 (pmIL-12) into BALB/c mice bearing subcutaneous tumors. Poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA) was used for complex formation with pmIL-12 in presence of 5% (w/v) glucose. Upon characterization, PAGA/pmIL-12 (3/1, ±) complexes were found to be most effective in gene transfer and were used consistently throughout this study. The levels of mIL-12 p70 and induced cytokines were determined by ELISA in the supernatant of the cultured tumors of the CT-26 subcutaneous tumor bearing BALB/c female mice 48 h after intratumoral injection of PAGA/pmIL-12 complexes and naked pmIL-12. The levels of IL-12, IFN-γ, TNF-α and NO were higher for the PAGA/pmIL-12 complexes than those for the naked pmIL-12, PAGA alone and 5% glucose injected groups. The relative presence of natural killer (NK) cells, CD4+ T cells, and antigen presenting cells, such as macrophages and dendritic cells determined using immunohistochemistry was higher for PAGA/pmIL-12 complexes compared with naked pmIL-12. The presence of CMV promoter in plasmid encoding IL-12 cDNAs did not induce any type I interferon response. There was a significant improvement in the survival rate and the inhibition of tumor growth after repeated injections of PAGA/pmIL-12 complexes.

Prevention of autoimmune insulitis by delivery of interleukin-4 plasmid using a soluble and biodegradable polymeric carrier

AbstractPurpose. We delivered interleukin-4 (IL-4) plasmid (pCAGGS-IL-4) using the biodegradable polymer, poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), to prevent autoimmune insulitis in NOD mice. Methods. The pCAGGS-IL-4/PAGA complex was transfected to 293T cells. The expression level of IL-4 was measured by ELISA. The pCAGGS IL-4/PAGA complex was injected once to NOD mice intravenously at the age of 4 weeks. RT-PCR was performed to evaluate the level of the IL-4 mRNA in the liver. At 6 weeks after the injection, the grade of insulitis of the mice was evaluated by double blind methods. Results.In vitro transfecton assays showed that PAGA enhanced the expression of IL-4 in 293T cells. RT-PCR of the liver showed that IL-4 was expressed highest in the complex injected group. In the plasmid/PAGA complex injected group, the prevalence of severe insulitis in NOD mice was markedly improved, suggesting that PAGA enhanced the delivery of IL-4 plasmid. Conclusion. The pCAGGS-IL-4/PAGA complex is an effective system to prevent autoimmune insulitis in NOD mice and applicable for the prevention of autoimmune diabetes.