Prevention of autoimmune insulitis by delivery of a chimeric plasmid encoding interleukin-4 and interleukin-10.

The combined administration of interleukin-4 (IL-4) and interleukin-10 (IL-10) expression plasmids has demonstrated synergistic effects on the prevention of autoimmune diabetes. To this end, we constructed a co-expression 'chimeric' plasmid, pCMV-IL4-IL10, in which the expression of IL-4 and IL-10 was driven by two separate CMV immediate early promoters by using the biodegradable polymer, poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) as a gene carrier to optimize gene delivery. In vitro transfection assays of the chimeric plasmid in 293T cells showed higher expression levels as well as dose dependence than the single gene expression plasmids. To evaluate the in vivo efficacy of the chimeric plasmid, the pCMV-IL4-IL10/PAGA complex was intravenously injected into 4-week-old non-obese diabetic (NOD) mice and compared to the co-administration group. While both groups had persistent gene expression longer than 5 weeks, the IL-4 and IL-10 serum levels of the chimeric group were higher than those in the co-administration group. Furthermore, the degree of insulitis in the chimeric group was improved over both the co-administration and non-injected control groups. These results suggest that the chimeric IL-4 and IL-10 expression plasmid can effectively reduce the incidence of autoimmune insulitis.     

Anti-GAD antibody targeted non-viral gene delivery to islet beta cells.

An islet cell targeting polymeric gene carrier was synthesized by conjugating anti-GAD Fab' fragment to PEI via PEG linker (PEI-PEG-Fab'). The Fab' fragment was prepared from a murine monoclonal antibody against glutamic acid decarboxylase (GAD), which has been identified as one of the major auto-antigens expressed in islet cells, and used as a targeting moiety for islet cell targeting. The electrophoretic migration of plasmid DNA (pCMVLuc)/PEI-PEG-Fab' complexes in agarose gel was completely retarded above the N/P ratio of 2. The complexes demonstrated a size of 100-275 nm with an almost neutral surface charge. Confocal microscopy revealed that the PEI-PEG-Fab' complexes showed much higher cellular binding and uptake efficiency compared to PEI-PEG complexes. The PEI-PEG-Fab' showed about 10-fold higher transfection efficiency (relative luciferase activity) than PEI-PEG in GAD-expressing mouse insulinoma cells (MIN6), however the transfection efficiency of PEI-PEG-Fab' reduced to that of PEI-PEG in GAD negative cells (293) and in the presence of competitive free Fab'. Considering the neutral surface charge of its complexes with DNA, and selectivity toward the islet cells expressing a specific antigen, the PEI-PEG-Fab' conjugate could be thought as a potential candidate of the systemic gene therapy for the treatment of type I diabetes.     

Deoxycholic acid-conjugated chitosan oligosaccharide nanoparticles for efficient gene carrier.

To develop chitosan based efficient gene carriers, highly purified chitosan oligosaccharides (COSs) were chemically modified with deoxycholic acid (DOCA). Owing to the amphiphilic characters, the DOCA-conjugated COSs (COSDs) formed self-aggregated nanoparticles in aqueous milieu. The physicochemical characterization revealed that the particle size of the nanoparticles was in the range of 200 approximately 240 nm and the critical aggregation concentration (cacs) was 0.012 approximately 0.046 g/L, depending on the degree of substitution (DS). As efficient gene carriers, the COSD nanoparticles showed superior gene condensation and protection of condensed gene from endonuclease attack than unmodified COSs. Furthermore, COSDs showed great potential for gene carrier with the high level of gene transfection efficiencies, even in the presence of serum. Considered with the negligible cytotoxic effects, DOCA-modified chitosan oligosaccharides can be considered as potential candidates for efficient non-viral gene carriers.     

Augmentation of erythropoietin enhancer-mediated hypoxia-inducible gene expression by co-transfection of a plasmid encoding hypoxia-inducible factor 1α for ischemic tissue targeting gene therapy

Therapeutic angiogenesis with gene encoding vascular endothelial growth factor (VEGF) is a potential treatment for ischemic diseases. However, VEGF expression should be tightly regulated to avoid side effects such as tumor growth. Previously, our group developed the erythropoietin (Epo) enhancer–SV40 promoter system for hypoxia-specific gene expression. In the present study, the activity of the Epo enhancer–SV40 promoter system was further enhanced without significant decrease in its specificity by co-transfection of the hypoxia-inducible factor 1α (HIF1α) gene. pSV-HIF1α was constructed by the insertion of the HIF1α cDNA into pSI. At a 1:1 ratio, co-transfection of pSV-HIF1α and pEpo-SV-Luc increased the promoter activity of the Epo enhancer–SV40 promoter system, showing at least three times higher gene expression under hypoxia as compared with the pEpo-SV-Luc single-plasmid transfection. Furthermore, co-transfection showed significant hypoxia specificity. Also, co-transfection of pEpo-SV-VEGF with pSV-HIF1α showed the enhanced VEGF expression without loss of hypoxia specificity, as compared with pEpo-SV-VEGF single-plasmid transfection. Furthermore, pSV-HIF1α induced the endogenous hypoxia-responsive genes such as angiopoietin-1, which would be beneficial for therapeutic angiogenesis. Therefore, with hypoxia specificity and higher gene expression, co-transfection of pSV-HIF1α and pEpo-SV-VEGF may be useful for ischemia targeting gene therapy.     

Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs

Polymeric nanosphere-mediated gene delivery may sustain the duration of plasmid DNA (pDNA) administration. In this study, poly(lactic-co-glycolic acid) (PLGA) nanospheres were evaluated as a gene carrier. The pDNA-loaded PLGA nanospheres were formulated with high encapsulation efficiency (87%). The nanospheres sustained release of pDNA for 11 days. The released pDNA maintained its structural and functional integrity. Furthermore, the PLGA nanospheres showed lower cytotoxicity than polyethylenimine (PEI) in vitro and in vivo. The nanospheres with vascular endothelial growth factor (VEGF) gene were injected into skeletal muscle of ischemic limb model, and gene expression mediated by the PLGA nanospheres with VEGF gene was compared to that of PEI/pDNA or naked pDNA in vivo. PLGA nanosphere/pDNA had significantly higher VEGF expression levels in comparison to PEI/pDNA and naked pDNA at 12 days after administration. In addition, gene therapy using PLGA nanospheres resulted in more extensive neovascularization at ischemic sites than both naked pDNA and PEI/pDNA. These results indicated that PLGA nanosphere might be useful as a potential carrier for skeletal muscle gene delivery applications.     

Dexamethasone conjugated poly(amidoamine) dendrimer as a gene carrier for efficient nuclear translocation

Nuclear membrane is one of the main barriers in polymer-mediated intracellular gene delivery. It was previously reported that glucocorticoid receptor dilated the nuclear pore and translocated into nucleus when it bound to its ligand, glucocorticoid. This suggests that the transport of DNA into nucleus may be facilitated by glucocorticoid. In this study, a glucocorticoid, dexamethasone, was conjugated to polyamidoamine (PAMAM) dendrimer and the effect of the conjugation was investigated. The PAMAM-Dexamethasone (PAM-Dexa) was synthesized by the one-step reaction using Traut's reagent. PAM-Dexa/plasmid DNA complex was completely retarded at a 1/1 weight ratio (polymer/DNA) in a gel retardation assay. PAM-Dexa protected DNA from DNase I for more than 60 min. PAM-Dexa/plasmid DNA complex showed the highest transfection efficiency to 293 cells at a 0.8/1 weight ratio. At this ratio, PAM-Dexa had higher transfection efficiency than PAMAM. Especially in the presence of serum during the transfection, the transfection efficiency of PAM-Dexa was higher than that of PAMAM or PEI by one order of magnitude. In addition, more PAM-Dexa/DNA complexes were observed in the nucleus region than PAMAM/DNA from the confocal microscopy studies. These results indicated that the technique with dexamethasone might be useful for the gene delivery using polymeric gene carriers and the development of efficient polymer vectors.     

Hypoxia-preconditioned adipose tissue-derived mesenchymal stem cell increase the survival and gene expression of engineered neural stem cells in a spinal cord injury model

Hypoxic preconditioning (HP) is a novel strategy to make stem cells resistant to the ischemic environment they encounter after transplantation into injured tissue; this strategy improves survival of both the transplanted cells and the host cells at the injury site. Using both in vitro and in vivo injury models, we confirmed that HP-treated adipose tissue-derived mesenchymal stem cells (HP-AT-MSCs) increased cell survival and enhanced the expression of marker genes in DsRed-engineered neural stem cells (NSCs-DsRed). Similar to untreated AT-MSCs, HP-AT-MSCs had normal morphology and were positive for the cell surface markers CD90, CD105, and CD29, but not CD31. In three in vitro ischemic-mimicking injury models, HP-AT-MSCs significantly increased both the viability of NSCs-DsRed and the expression of DsRed and clearly reduced the number of annexin-V-positive apoptotic NSCs-DsRed and the expression of the apoptotic factor Bax. Consistent with the in vitro assay, co-transplantation of NSCs-DsRed with HP-AT-MSCs significantly improved the survival of the NSCs-DsRed, resulting in an increased expression of the DsRed reporter gene at the transplantation site in a rat spinal cord injury (SCI) model. These findings suggest that the co-transplantation of HP-AT-MSCs with engineered NSCs can improve both the cell survival and the gene expression of the engineered NSCs, indicating that this novel strategy can be used to augment the therapeutic efficacy of combined stem cell and gene therapies for SCI.     

Therapeutic use of H2O2-responsive anti-oxidant polymer nanoparticles for doxorubicin-induced cardiomyopathy

Doxorubicin (DOX) is a commonly used anti-neoplastic agent but its clinical use is limited due to serious hepatic and cardiac side effects. DOX-induced toxicity is mainly associated with overproduction of reactive species oxygen (ROS) such as hydrogen peroxide (H₂O₂). We have recently developed H₂O₂-responsive anti-oxidant polymer, polyoxalate containing vanillyl alcohol (PVAX), which is designed to rapidly scavenge H₂O₂ and release vanillyl alcohol with anti-oxidant, anti-inflammatory and anti-apoptotic properties. In this study, we report that PVAX nanoparticles are novel therapeutic agents for treating DOX-induced cardiac and hepatic toxicity. Intraperitoneal injection of PVAX nanoparticles (4 mg/kg/day) resulted in significant inhibition in apoptosis in liver and heart of DOX-treated mice by suppressing the activation of poly (ADP ribose) polymerase 1 (PARP-1) and caspase-3. PVAX treatment also prevented DOX-induced cardiac dysfunction. Furthermore, survival rate (vehicle = 35% vs. PVAX = 75%; p < 0.05) was significantly improved in a PVAX nanoparticles-treated group compared with vehicle treated groups. Taken together, we anticipate that PVAX nanoparticles could be a highly specific and potent treatment modality in DOX-induced cardiac and hepatic toxicity.