Eunmi Kim

Drawing Sticky Adeno‐Associated Viruses on Surfaces for Spatially Patterned Gene Expression

In developing tissues, the spatially controlled secretion of extracellular signals creates biointerfaces that modulate cellular processes of differentiation, proliferation, and migration, and provide molecular cues to organize the structure of tissues. Systems that control spatial distribution of extracellular molecules on substrates have been developed in order to induce patterned expression of intracellular inductive factors. 5,6] Alternatively, spatially patterned gene delivery has been employed to overcome limitations found in protein delivery: short protein half-life and systemic toxicity. The development of in vitro model systems that mimic the spatial control of gene expression in tissues or organs is critical to elucidate a variety of biological mechanisms. However, the majority of gene delivery systems rely on simple additions of gene carriers directly to media, which are inherently limited in their ability to spatially control gene expression. Advances in microand nanofabrication technologies have enabled researchers to control locations of gene vectors on surfaces. 11] Existing methods include microfluidics, surface coating, and self-assembly. In general, the techniques involve multiple laborious procedures, typically chemical activation of substrates, resist depositions, pattern generation using a photomask, and gene vector immobilization. Furthermore, expensive equipment is often necessary. Unlike the aforementioned complex processes, this study describes a simple, versatile approach for spatially patterned gene delivery inspired by adhesion of marine mussels. Catecholamine, the key adhesive moiety found in the specialized adhesive proteins of mytilus edulis, was used to formulate “sticky” viruses. The adhesive catecholamine polymer used in this study is poly(ethylenimine)-catechol (PEI-C), which has been used for material-independent layerby-layer assembly and mechanical reinforcement of carbon nanotube fibers. The adeno-associated virus (AAV), which is a safe and efficient parvovirus, was complexed with the PEIC. Because of the underwater adhesive property of PEI-C, the AAV vector complexed with the PEI-C became a highly sticky virus that can stably adhere onto surfaces. Most importantly, by using the sticky viral vectors, we were able to use a micropipette as a “pen” to create viral patterns on substrates. This “genevector drawing” technique bypasses laborious multiple steps and can thus be a versatile platform to control gene expression for the establishment of complex tissues. Branched PEI was conjugated with 3-(3,4-dihydroxyphenyl) propionic acid (DPA) to generate PEI-C, which provides sticky viral vectors (Figure 1A). A novel virus, AAVr3.45, which was specifically designed for neuronal cell

Heparin-coated superparamagnetic nanoparticle-mediated adeno-associated virus delivery for enhancing cellular transduction

Superparamagnetic iron oxide nanoparticles (SPIONs) have been exploited as an elegant vehicle to enhance gene delivery efficiencies in gene therapy applications. We developed a magnetically guided adeno-associated virus (AAV) delivery system for enhancing gene delivery to HEK293T and PC12 cell lines. Wild-type AAV2 and a novel AAV vector, AAVr3.45, which was directly evolved in a previous study to possess diverse cell tropisms, were used as gene carriers. Additionally, the affinity of each viral vector to heparin was employed as a moiety to immobilize virus onto heparin-coated SPIONs (HpNPs). Magnetically guided AAV delivery resulted fast and efficient cellular transduction. Importantly, a short exposure of virus to target cells under a magnetic field (<180min) yielded comparable transduction produced by the conventional gene-delivery protocol (i.e., 24h-incubation of virus with target cells prior to replacing with fresh medium). Additionally, magnetic guidance of AAV encoding nerve growth factor (NGF) produced sufficient functional NGF, leading to robust neurite elongation by PC12 as compared to direct NGF protein delivery or non-magnetic delivery. The successful establishment of a magnetically guided AAV delivery system, with the ability to efficiently and rapidly infect target cells, will provide a powerful platform for a variety of gene therapy applications.

A new adaptive fuzzy controller using the parallel structure of fuzzy controller and its application

Abstract The paper suggests a new fuzzy adaptive controller, which is able to solve the problems of classical adaptive controllers and conventional fuzzy adaptive controllers. It explains the architecture of a fuzzy adaptive controller using the robust property of a fuzzy controller. We propose a design procedure which can be carried out mathematically and systematically from the model of a controlled system as well as related mathematical theorems and their proofs are also given. The performance of the proposed adaptive control algorithm is analyzed through a design example and an experiment on hybrid force/position robot control system.

Magnetically enhanced adeno-associated viral vector delivery for human neural stem cell infection

Gene therapy technology is a powerful tool to elucidate the molecular cues that precisely regulate stem cell fates, but developing safe vehicles or mechanisms that are capable of delivering genes to stem cells with high efficiency remains a challenge. In this study, we developed a magnetically guided adeno-associated virus (AAV) delivery system for gene delivery to human neural stem cells (hNSCs). Magnetically guided AAV delivery resulted in rapid accumulation of vectors on target cells followed by forced penetration of the vectors across the plasma membrane, ultimately leading to fast and efficient cellular transduction. To combine AAV vectors with the magnetically guided delivery, AAV was genetically modified to display hexa-histidine (6xHis) on the physically exposed loop of the AAV2 capsid (6xHis AAV), which interacted with nickel ions chelated on NTA-biotin conjugated to streptavidin-coated superparamagnetic iron oxide nanoparticles (NiStNPs). NiStNP-mediated 6xHis AAV delivery under magnetic fields led to significantly enhanced cellular transduction in a non-permissive cell type (i.e., hNSCs). In addition, this delivery method reduced the viral exposure times required to induce a high level of transduction by as much as to 2-10 min of hNSC infection, thus demonstrating the great potential of magnetically guided AAV delivery for numerous gene therapy and stem cell applications.

Transforming growth factor-β gene silencing using adenovirus expressing TGF-β1 or TGF-β2 shRNA

Tumor cells secrete a variety of cytokines to outgrow and evade host immune surveillance. In this context, transforming growth factor-β1 (TGF-β1) is an extremely interesting cytokine because it has biphasic effects in cancer cells and normal cells. TGF-β1 acts as a growth inhibitor in normal cells, whereas it promotes tumor growth and progression in tumor cells. Overexpression of TGF-β1 in tumor cells also provides additional oncogenic activities by circumventing the host immune surveillance. Therefore, this study ultimately aimed to test the hypothesis that suppression of TGF-β1 in tumor cells by RNA interference can have antitumorigenic effects. However, we demonstrated here that the interrelation between TGF-β isotypes should be carefully considered for the antitumor effect in addition to the selection of target sequences with highest efficacy. The target sequences were proven to be highly specific and effective for suppressing both TGF-β1 mRNA and protein expression in cells after infection with an adenovirus expressing TGF-β1 short hairpin RNA (shRNA). A single base pair change in the shRNA sequence completely abrogated the suppressive effect on TGF-β1. Surprisingly, the suppression of TGF-β1 induced TGF-β3 upregulation, and the suppression of TGF-β2 induced another unexpected downregulation of both TGF-β1 and TGF-β3. Taken together, this information may prove useful when considering the design for a novel cancer immunogene therapy.

The promotion of human neural stem cells adhesion using bioinspired poly(norepinephrine) nanoscale coating

The establishment of versatile biomaterial interfaces that can facilitate cellular adhesion is crucial for elucidating the cellular processes that occur on biomaterial surfaces. Furthermore, biomaterial interfaces can provide physical or chemical cues that are capable of stimulating cellular behaviors by regulating intracellular signaling cascades. Herein, a method of creating a biomimetic functional biointerface was introduced to enhance human neural stem cell (hNSC) adhesion. The hNSC-compatible biointerface was prepared by the oxidative polymerization of the neurotransmitter norepinephrine, which generates a nanoscale organic thin layer, termed poly(norepinephrine) (pNE). Due to its adhesive property, pNE resulted in an adherent layer on various substrates, and pNE-coated biointerfaces provided a highly favorable microenvironment for hNSCs, with no observed cytotoxicity. Only a 2-hour incubation of hNSCs was required to firmly attach the stem cells, regardless of the type of substrate. Importantly, the adhesive properties of pNE interfaces led to micropatterns of cellular attachment, thereby demonstrating the ability of the interface to organize the stem cells. This highly facile surface-modification method using a biomimetic pNE thin layer can be applied to a number of suitable materials that were previously not compatible with hNSC technology.

Integration of adeno-associated virus-derived peptides into nonviral vectors to synergistically enhance cellular transfection.

This study describes a simple, versatile approach for developing a nonviral gene carrier by adopting the highly efficient gene delivery properties of the adeno-associated virus (AAV). Specific viral peptides (r3.45_hepBD) extracted from AAV r3.45, which directly evolved to improve gene delivery capabilities in many cell types, were conjugated onto branched polyethylenimine (PEI) to form hybrid gene carriers. AAV r3.45 carries a sequence insertion (LATQVGQKTA; r3.45) within the heparin-binding domain (LQRGNRQA; hepBD), which ultimately comprises a novel sequence (LQRGNLATQVGQKTARQA; r3.45_hepBD) on the capsid. This sequence is hypothesized to be a crucial cue to enhance gene delivery efficiency. Consequently, the intimate interactions of the conjugated r3.45_hepBD with the glycosaminoglycans, including chondroitin sulfate, resulted in significantly enhanced cellular transfection of DNA/PEI-r3.45_hepBD complexes. The successful establishment of a nonviral system that is built with novel peptides will provide a powerful means for developing a substantial number of gene therapy applications.

Assessment of cerebral oxygen supply-demand balance by near-infrared spectroscopy during induction of anesthesia in patients undergoing coronary artery bypass graft surgery: comparison of midazolam with propofol

BACKGROUND Near-infrared spectroscopy (NIRS) continuously measures regional cerebral oxygen saturation (rSO2) noninvasively and has been shown to detect even small changes in cerebral oxygen supply-demand balance. Although widely used, only the effect of midazolam on cerebral blood flow has been studied in humans and evidence is lacking about its effect on cerebral metabolic rate. We therefore evaluated the effect of midazolam on cerebral oxygen supply-demand balance with NIRS. METHODS Sixty patients undergoing elective coronary artery bypass graft surgery were randomly allocated into either midazolam (n = 30) or propofol (n = 30) group. rSO2 was recorded before induction while patients were breathing room air as baseline, after pre-oxygenation with 100% oxygen, after administration of either midazolam or propofol, after completion of administration of sufentanil and after tracheal intubation. Hemodynamic variables including cardiac index and mixed venous oxygen saturation were recorded at the same time points. RESULTS rSO2 and hemodynamic variables were similar between the groups throughout the study period. After pre-oxygenation, rSO2 significantly increased compared to baseline in each group, and did not show any additional increase after administration of either midazolam or propofol and sufentanil in both groups. CONCLUSIONS Midazolam preserves cerebral blood flow-metabolism coupling to a similar degree to propofol as assessed by near infrared spectroscopy.

Transduction of striatum and cortex tissues by adeno-associated viral vectors produced by herpes simplex virus- and baculovirus-based methods

Recombinant adeno-associated virus (AAV) vectors can be engineered to carry genetic material encoding therapeutic gene products that have demonstrated significant clinical promise. These viral vectors are typically produced in mammalian cells by the transient transfection of two or three plasmids encoding the AAV rep and cap genes, the adenovirus helper gene, and a gene of interest. Although this method can produce high-quality AAV vectors when used with multiple purification protocols, one critical limitation is the difficulty in scaling-up manufacturing, which poses a significant hurdle to the broad clinical utilization of AAV vectors. To address this challenge, recombinant herpes simplex virus type I (rHSV-1)- and recombinant baculovirus (rBac)-based methods have been established recently. These methods are more amenable to large-scale production of AAV vectors than methods using the transient transfection of mammalian cells. To investigate potential applications of AAV vectors produced by rHSV-1- or rBac-based platforms, the in vivo transduction of rHSV-1- or rBac-produced AAV serotype 2 (AAV2) vectors within the rat brain were examined by comparing them with vectors generated by the conventional transfection method. Injection of rHSV-1- or rBac-produced AAV vectors into rat striatum and cortex tissues revealed no differences in cellular tropism (i.e., predominantly neuronal targeting) or anteroposterior spread compared with AAV2 vectors produced by transient transfection. This report represents a step towards validating AAV vectors produced by the rHSV-1- and the rBac-based systems as promising tools, especially for delivering therapeutic molecules to the central nervous system.

Accuracy of the epidural catheter position during the lumbar approach in infants and children: a comparison among L2-3, L3-4, and L4-5 approaches

Background The aim of this study was to compare the accuracy of the position of the epidural catheter inserted from three different lumbar intervertebral spaces, L2-3, L3-4, and L4-5, in infants and children. Methods Seventy-five children were randomly allocated to 3 groups according to the epidural catheter insertion site (L2-3, L3-4, and L4-5). The epidural catheter tip was identified using 50% diluted Iohexol and fluoroscopy. The incidence of correct position was compared among the groups and between infants and children. Results The incidence of correct position was significantly higher in the L2-3 group as compared to the L3-4 and L4-5 groups (P = 0.023 and P = 0.046 respectively). The incidence of correct position was higher in infants compared to children (P = 0.017). Conclusions The L2-3 intervertebral space is preferable during epidural catheter insertion in children older than 1 year, but a low lumbar level should be considered in infants because they have a higher risk of neural damage.