Arginine-grafted bioreducible poly(disulfide amine) for gene delivery systems

Arginine-grafted bioreducible poly(disulfide amine) (ABP) polymer was synthesized for non-viral gene delivery systems. Its Mw was measured to be 4.45 × 10³ Da/mole by FPLC-SEC and its PDI value was 1.49. ABP was able to retard pDNA from a weight ratio of 2 but ABP could not retard pDNA even at a weight ratio of 10 in the presence of DTT, showing that it can be biodegraded in reducing environment such as cytoplasm. ABP was examined to form positively charged nano-sized particles (<200 nm) with pDNA. ABP showed no significant cytotoxicity and greatly enhanced transfection efficiency in comparison with unmodified poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH)) and PEI25k in mammalian cells. The transfection efficiency of ABP was not much reduced even in the serum condition. Chloroquine treatment was not found to improve the transfection efficiency of ABP. The cellular uptake pattern of ABP polyplexes was almost similar with poly(CBA-DAH), suggesting that greatly enhanced transfection efficiency of ABP is not induced by its high cellular penetrating ability but may be mediated by other factors such as good nuclear localization ability.     

Water-Soluble and Low Molecular Weight Chitosan-Based Plasmid DNA Delivery

Purpose. Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery because of its high positive charges and low cytotoxicity. In this study, low molecular weight chitosan (LMWC, molecular weight of 22 kDa) was characterized and evaluated as a gene carrier. Methods. Plasmid/LMWC complex was analyzed in 1% agarose gel electrophoresis. To confirm that the LMWC protected plasmids from nuclease, DNase I protection assays were performed. pSV--galactosidase plasmid/LMWC complex was transfected into 293T cells and transfection efficiency was evaluated by -galactosidase assay. Cytotoxicity of LMWC was determined by MTT assay. Results. Unlike high molecular weight chitosan (HMWC), LMWC is highly water soluble, and can form complex with plasmids in physiological buffer. The plasmid DNA was completely retarded at a weight ratio of 1:2 (plasmid:LMWC) in 1% agarose gel. DNase I protection assay showed that plasmids were protected from DNase I over 60 min. The most efficient transfection was obtained at a weight ratio of 1:3 (plasmid:LMWC). The transfection efficiency of LMWC was significantly higher than naked DNA and higher than poly-L-lysine (PLL). MTT assay showed that LMWC was less cytotoxic than PLL. Conclusions. LMWC is non-toxic and has higher transfection efficiency than PLL. Therefore, LMWC will be useful in the development of safe gene carriers.     

Polymeric gene carrier for insulin secreting cells: poly(L-lysine)-g-sulfonylurea for receptor mediated transfection.

Ex vivo transfer of therapeutic genes to cells is one of the potential strategies to prolong the life span of cell transplants. However, relatively safe non-viral carriers have not been extensively investigated due to their lower transfection efficiency. In this study, poly(L-lysine)-g-sulfonylurea varying SU content (PLL-SU) was synthesized to promote gene delivery efficacy to an insulin secreting cell line, RINm5F, which is known to express sulfonylurea receptor (SUR). The polymer formed complexes with a model reporter gene of pCMV-Luc (DNA) and the size of resulting particles was around 100 nm. The transfection efficiency of a polymer synthesized with 5 mol% of SU in the reaction feed (PLL-SU5%) to RINm5F cell was at least 5 times higher than that of PLL. The cytotoxicity of PLL-SU5%/DNA complex was equivalent to that of PLL/DNA complex. PLL-SU5% showed less transfection efficiency than PLL to NIH3T3 and HepG2 cells which are SUR negative. In RINm5F cells, the addition of free SU decreased the transfection efficiency of PLL-SU5%/DNA complex, suggesting that the complex shares the same receptors for SU. The PLL-SU5%/DNA complex seems to be internalized via SUR-mediated endocytosis pathway as suggested by vacuolar ATPases inhibition by Bafilomycin A1. It is noted that RINm5F cells treated with PLL-SU5%/DNA complex secreted more insulin than control, untreated cells, suggesting the insulinotropic effect of SU in PLL-SU5%. In conclusion, PLL-SU may be useful for transfer of therapeutic genes into insulin secreting cells.     

Hypoxia-inducible gene expression system using the erythropoietin enhancer and 3'-untranslated region for the VEGF gene therapy.

Gene therapy with the vascular endothelial growth factor (VEGF) gene is a potential treatment for many disorders or injuries with ischemia. However, unregulated expression of VEGF may induce pathological angiogenesis, promoting tumor growth, diabetic proliferative retinopathy and rupture of atherosclerotic plaque. Therefore, the effective regulation of the gene expression is one of the requirements for the VEGF gene therapy. In this research, we evaluated the hypoxia-inducible gene expression system with the erythropoietin (Epo) enhancer and the Epo 3'-untranslated region (UTR). The luciferase plasmids were constructed with the Epo enhancer (pEpo-SV-Luc), the Epo 3'-UTR (pSV-Luc-EpoUTR) or both (pEpo-SV-Luc-EpoUTR). The polyethylenimine/plasmid complexes were transfected to 293 or A7R5 cells and the cells were incubated under normoxia or hypoxia. The results showed that the Epo enhancer or Epo 3'-UTR increased the target gene expression under hypoxia. pEpo-SV-Luc-EpoUTR showed the highest luciferase expression. The VEGF expression plasmid with the Epo enhancer and 3'-UTR was also constructed. The VEGF expression by pEpo-SV-VEGF-EpoUTR showed the highest specificity of the gene expression in the hypoxic cells. The results suggest that the VEGF plasmid with the Epo enhancer and the Epo 3'-UTR may be useful for gene therapy for ischemic diseases.     

Suicide gene therapy using reducible poly (oligo-D-arginine) for the treatment of spinal cord tumors

Suicide gene therapy based on a combination of herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir (GCV) has obstacles to achieving a success in clinical use for the treatment of cancer due to inadequate thymidine kinase (TK) expression. The primary concern for improving anticancer efficacy of the suicide gene therapy is to develop an appropriate carrier that highly expresses TK in vivo. Despite great advances in the development of non-viral vectors, none has been used in cancer suicide gene therapy, not even in experimental challenge. Reducible poly (oligo-D-arginine) (rPOA), one of the effective non-viral carriers working in vivo, was chosen to deliver HSV-tk to spinal cord tumors which are appropriate targets for suicide gene therapy. Since the system exerts toxicity only in dividing cells, cells in the central nervous system, which are non-proliferative, are not sensitive to the toxic metabolites. In the present study, we demonstrated that the locomotor function of the model rat was maintained through the tumor suppression resulting from the tumor-selective suicide activity by co-administration of rPOA/HSV-tk and GCV. Thus, rPOA plays a crucial role in suicide gene therapy for cancer, and an rPOA/HSV-tk and GCV system could help promote in vivo trials of suicide gene therapy.     

A Gene and Neural Stem Cell Therapy Platform Based on Neuronal Cell Type-Inducible Gene Overexpression

Purpose

Spinal cord injury (SCI) is associated with permanent neurological damage, and treatment thereof with a single modality often does not provide sufficient therapeutic outcomes. Therefore, a strategy that combines two or more techniques might show better therapeutic effects.

Materials and Methods

In this study, we designed a combined treatment strategy based on neural stem cells (NSCs) introduced via a neuronal cell type-inducible transgene expression system (NSE::) controlled by a neuron-specific enolase (NSE) promoter to maximize therapeutic efficiency and neuronal differentiation. The luciferase gene was chosen to confirm whether this combined system was working properly prior to using a therapeutic gene. The luciferase expression levels of NSCs introduced via the neuronal cell type-inducible luciferase expression system (NSE::Luci) or via a general luciferase expressing system (SV::Luci) were measured and compared in vitro and in vivo.

Results

NSCs introduced via the neuronal cell type-inducible luciferase expressing system (NSE::Luci-NSCs) showed a high level of luciferase expression, compared to NSCs introduced via a general luciferase expressing system (SV::Luci-NSCs). Interestingly, the luciferase expression level of NSE::Luci-NSCs increased greatly after differentiation into neurons.

Conclusion

We demonstrated that a neuronal cell type-inducible gene expression system is suitable for introducing NSCs in combined treatment strategies. We suggest that the proposed strategy may be a promising tool for the treatment of neurodegenerative disorders, including SCI.     

Erythropoietin gene delivery using an arginine-grafted bioreducible polymer system

Erythropoietin (EPO) plays a key regulatory role in the formation of new red blood cells (RBCs). Erythropoietin may also have a role as a therapeutic agent to counteract ischemic injury in neural, cardiac and endothelial cells. One of the limitations preventing the therapeutic application of EPO is its short half-life. The goal of this study was to develop a gene delivery system for the prolonged and controlled release of EPO. The arginine grafted bioreducible polymer (ABP) and its PEGylated version, ABP-PEG10, were utilized to study the expression efficiency and therapeutic effectiveness of this erythropoietin gene delivery system in vitro. Poly(ethylene glycol) (PEG) modification of the ABP was employed to inhibit the particle aggregation resulting from the interactions between cationic polyplexes and the negatively charged proteins typically present in serum. Both the ABP and the ABP-PEG10 carriers demonstrated efficient transfection and long-term production of EPO in a variety of cell types. The expressed EPO protein stimulated hematopoietic progenitor cells to form significant numbers of cell colonies in vitro. These data confirm that this EPO gene delivery system using a bioreducible polymeric carrier, either ABP or ABP-PEG 10, merits further testing as a potential therapeutic modality for a variety of clinically important disease states.     

Modular hip exoskeleton improves walking function and reduces sedentary time in community-dwelling older adults

Numerous studies showed that postural balance improves through light touch on a stable surface highlighting the importance of haptic information, seemingly downplaying the mechanical contributions of the support. The present study examined the mechanical effects of canes for assisting balance in healthy individuals challenged by standing on a beam. Sixteen participants supported themselves with two canes, one in each hand, and applied minimal, preferred, or maximum force onto the canes. They positioned the canes in the frontal plane or in a tripod configuration. Statistical analysis used a linear mixed model to evaluate the effects on the center of pressure and the center of mass. The canes significantly reduced the variability of the center of pressure and the center of mass to the same level as when standing on the ground. Increasing the exerted force beyond the preferred level yielded no further benefits, although in the preferred force condition, participants exploited the altered mechanics by resting their arms on the canes. The tripod configuration allowed for larger variability of the center of pressure in the task-irrelevant anterior–posterior dimension. High forces had a destabilizing effect on the canes: the displacement of the hand on the cane handle increased with the force. Given this static instability, these results show that using canes can provide not only mechanical benefits but also challenges. From a control perspective, effort can be reduced by resting the arms on the canes and by channeling noise in the task-irrelevant dimensions. However, larger forces exerted onto the canes can also have destabilizing effects and the instability of the canes needs to be counteracted, possibly by arm and shoulder stiffness. Insights into the variety of mechanical effects is important for the design of canes and the instructions of how to use them.