Uptake Of Plasmid DNA Research Articles

Anti-inflammatory loaded poly-lactic glycolic acid nanoparticle formulations to enhance myocardial gene transfer: an in-vitro assessment of a drug/gene combination therapeutic approach for direct injection.

BackgroundCardiac gene therapy for heart disease is a major translational research area with potential, yet problems with safe and efficient gene transfer into cardiac muscle remain unresolved. Existing methodology to increase vector uptake include modifying the viral vector, non-viral particle encapsulation and or delivery with device systems. These advanced methods have made improvements, however fail to address the key problem of inflammation in the myocardium, which is known to reduce vector uptake and contribute to immunogenic adverse events. Here we propose an alternative method to co-deliver anti-inflammatory drugs in a controlled release polymer with gene product to improve therapeutic effects.MethodsA robust, double emulsion production process was developed to encapsulate drugs into nanoparticles. Briefly in this proof of concept study, aspirin and prednisolone anti-inflammatory drugs were encapsulated in various poly-lactic glycolic acid polymer (PLGA) formulations. The resultant particle systems were characterized, co-delivered with GFP plasmid, and evaluated in harvested myocytes in culture for uptake.ResultsHigh quality nanoparticles were harvested from multiple production runs, with an average 64 ± 10 mg yield. Four distinct particle drug system combinations were characterized and evaluated in vitro: PLGA(50:50) Aspirin, PLGA(65:35) Prednisolone, PLGA(65:35) Aspirin and PLGA(50:50) Prednisolone Particles consisted of spherical shape with a narrow size distribution 265 ± 104 nm as found in scanning electron microscopy imaging. Prednisolone particles regardless of PLGA type were found on average ≈ 100 nm smaller than the aspirin types. All four groups demonstrated high zeta potential stability and re-constitution testing prior to in vitro. In vitro results demonstrated co uptake of GFP plasmid (green) and drug loaded particles (red) in culture with no incidence of toxicity.ConclusionsNano formulated anti-inflammatories in combination with standalone gene product therapy may offer a clinical solution to maximize cardiac gene therapy product effects while minimizing the risk of the host response in the inflammatory myocardial environment.

Previously Undescribed Plasmids Recovered from Activated Sludge Confer Tetracycline Resistance and Phenotypic Changes to Acinetobacter oleivorans DR1

We used culture-dependent and culture-independent methods to extract previously undescribed plasmids harboring tetracycline (TC) resistance genes from activated sludge. The extracted plasmids were transformed into naturally competent Acinetobacter oleivorans DR1 to recover a non-Escherichia coli-based plasmid. The transformed cells showed 80-100-fold higher TC resistance than the wild-type strain. Restriction length polymorphism performed using 30 transformed cells showed four different types of plasmids. Illumina-based whole sequencing of the four plasmids identified three previously unreported plasmids and one previously reported plasmid. All plasmids carried TC resistance-related genes (tetL, tetH), tetracycline transcriptional regulators (tetR), and mobilization-related genes. As per expression analysis, TC resistance genes were functional in the presence of TC. The recovered plasmids showed mosaic gene acquisition through horizontal gene transfer. Membrane fluidity, hydrophobicity, biofilm formation, motility, growth rate, sensitivity to stresses, and quorum sensing signals of the transformed cells were different from those of the wild-type cells. Plasmid-bearing cells seemed to have an energy burden for maintaining and expressing plasmid genes. Our data showed that acquisition of TC resistance through plasmid uptake is related to loss of biological fitness. Thus, cells acquiring antibiotic resistance plasmids can survive in the presence of antibiotics, but must pay ecological costs.

A Ternary Conjugation System for the Construction of DNA Libraries forGeobacillus kaustophilusHTA426

This report describes efficient plasmid uptake by the thermophile Geobacillus kaustophilus HTA426 by means of a ternary conjugation system, which was used to construct thermophile DNA libraries for G. kaustophilus and to identify the genes for orotidine-5'-phosphate decarboxylase by in vivo functional screening. The results indicate that the conjugation system is useful in constructing G. kaustophilus libraries, which are practical in identifying thermophile genes.

Impact of Plasmid Quality on Lipoplex-Mediated Transfection

This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (-46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine®, this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials.

Efficacy and mechanism of poloxamine‐assisted polyplex transfection

Amphiphilic block copolymers acting as biological response modifiers provide an attractive approach for improving the transfection efficiency of polycationic polymer/DNA complexes (polyplexes) by altering cellular processes crucial for efficient transgene expression. The present study aimed to investigate the effect of the poloxamine Tetronic T904, a four-arm polyethylene oxide/polypropylene oxide block copolymer, on polyplex transfection and to determine its mechanism of action by analyzing the cellular uptake of polyplex, the nuclear localization of plasmid and RNA transcript production. T904 significantly increased the transfection efficiency of polyplexes based on 25-kDa branched polyethylenimine in a dose-dependent manner in the presence of serum in C6 glioma cells, as well as human fibroblasts and mesenchymal stem cells. The activity of T904 was not promoter-dependent, increasing the expression of reporter genes under both cytomegalovirus and SV40 promoters. Although T904 did not affect the internalization or nuclear uptake of plasmid, mRNA expression levels from both promoters showed dose-dependent increases that closely paralleled increases in gene expression. The present study demonstrates that T904 significantly increases polyplex transfection efficiency and suggests a mechanism of increased transcriptional activity. As a four-arm, hydroxyl-terminated polymer, T904 is amenable to a variety of end group functionalization and covalent cross-linking strategies that have been developed for preparing hydrogels from multi-arm polyethylene glycol, making it particularly attractive for scaffold-mediated gene delivery.

Nanosecond Electric Pulse Effects on Gene Expression

Gene electrotransfection using micro- or millisecond electric pulses is a well-established method for safe gene transfer. For efficient transfection, plasmid DNA has to reach the nucleus. Shorter, high-intensity nanosecond electric pulses (nsEPs) affect internal cell membranes and may contribute to an increased uptake of plasmid by the nucleus. In our study, nsEPs were applied to Chinese hamster ovary (CHO) cells after classical gene electrotransfer, using micro- or millisecond pulses with a plasmid coding the green fluorescent protein (GFP). Time gaps between classical gene electrotransfer and nsEPs were varied (0.5, 2, 6 and 24 h) and three different nsEP parameters were used: 18 ns-10 kV/cm, 10 ns-40 kV/cm and 15 ns-60 kV/cm. Results analyzed by either fluorescence microscopy or flow cytometry showed that neither the percentage of electrotransfected cells nor the amount of GFP expressed was increased by nsEP. All nsEP parameters also had no effects on GFP fluorescence intensity of human colorectal tumor cells (HCT-116) with constitutive expression of GFP. We thus conclude that nsEPs have no major contribution to gene electrotransfer in CHO cells and no effect on constitutive GFP expression in HCT-116 cells.

The broader context of antibiotic resistance: Zinc feed supplementation of piglets increases the proportion of multi-resistant Escherichia coli in vivo

Following the Europe-wide ban of antimicrobial growth promoters, feed supplementation with zinc has increased in livestock breeding. In addition to possible beneficial effects on animal health, feed supplementation with heavy metals is known to influence the gut microbiota and might promote the spread of antimicrobial resistance via co-selection or other mechanisms. As Escherichia coli is among the most important pathogens in pig production and often displays multi-resistant phenotypes, we set out to investigate the influence of zinc feed additives on the composition of the E. coli populations in vivo focusing on phylogenetic diversity and antimicrobial resistance.In a piglet feeding trial, E. coli were isolated from ileum and colon digesta of high dose zinc-supplemented (2500ppm) and background dose (50ppm) piglets (control group). The E. coli population was characterized via pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) for the determination of the phylogenetic background. Phenotypic resistance screening via agar disk diffusion and minimum inhibitory concentration testing was followed by detection of resistance genes for selected clones.We observed a higher diversity of E. coli clones in animals supplemented with zinc compared to the background control group. The proportion of multi-resistant E. coli was significantly increased in the zinc group compared to the control group (18.6% vs. 0%).For several subclones present both in the feeding and the control group we detected up to three additional phenotypic and genotypic resistances in the subclones from the zinc feeding group. Characterization of these subclones suggests an increase in antimicrobial resistance due to influences on plasmid uptake by zinc supplementation, questioning the reasonability of zinc feed additives as a result of the ban of antimicrobial growth promoters.

Gene delivery into human cancer cells by cationic lipid-mediated magnetofection

In this study, a combination of magnetic nanoparticles (MNPs) together with cationic lipid N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium chloride formulated with colipid cholesterol, upon magnetofection, enhanced DNA uptake into human glioblastoma-astrocytoma, epithelial-like cell line U-87 MG, hepatocellular carcinoma Hep G2, cervical cancer HeLa and breast cancer MDA-MB-231 cells. Having confirmed this, we monitored uptake of plasmid DNA mediated by ternary magnetoplexes by fluorescence microscopy, flow cytometry and reporter gene expression assays in the presence and absence of a magnetic field. Our observations clearly indicate enhanced transfection efficiency in vitro, upon magnetofection, in the presence of serum as seen from β-Gal reporter gene expression. The observed activity in serum suggests the suitability of MNPs for in vivo applications. Further, we measured the transverse relaxation time (T2) and obtained T2-weighted MRI images of treated U-87 MG cells. T2 determined for MNP-VP-Me22 and MNP-VP-Et22 corresponds to 22.6±0.8 ms and 36.0±2.1 ms, respectively, as compared to 47±1.7 ms for control, suggesting their applicability in molecular imaging. Our results collectively highlight the potential of lipid-based approach to augment magnetic-field guided-gene delivery using MNPs and additionally towards developing intracellular molecular probes for magnetic resonance imaging.

Direct Head-To-Head Comparison of Cationic Liposome-Mediated Gene Delivery to Mesenchymal Stem/Stromal Cells of Different Human Sources: A Comprehensive Study

Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources-bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP(+) cells 48 hr post-transfection, 58 ± 7.1% and 54 ± 3.8%, respectively, versus 33 ± 4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP(+) cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.

Micro-/nanofluidics based cell electroporation.

Non-viral gene delivery has been extensively explored as the replacement for viral systems. Among various non-viral approaches, electroporation has gained increasing attention because of its easy operation and no restrictions on probe or cell type. Several effective systems are now available on the market with reasonably good gene delivery performance. To facilitate broader biological and medical applications, micro-/nanofluidics based technologies were introduced in cell electroporation during the past two decades and their advances are summarized in this perspective. Compared to the commercially available bulk electroporation systems, they offer several advantages, namely, (1) sufficiently high pulse strength generated by a very low potential difference, (2) conveniently concentrating, trapping, and regulating the position and concentration of cells and probes, (3) real-time monitoring the intracellular trafficking at single cell level, and (4) flexibility on cells to be transfected (from single cell to large scale cell population). Some of the micro-devices focus on cell lysis or fusion as well as the analysis of cellular properties or intracellular contents, while others are designed for gene transfection. The uptake of small molecules (e.g., dyes), DNA plasmids, interfering RNAs, and nanoparticles has been broadly examined on different types of mammalian cells, yeast, and bacteria. A great deal of progress has been made with a variety of new micro-/nanofluidic designs to address challenges such as electrochemical reactions including water electrolysis, gas bubble formation, waste of expensive reagents, poor cell viability, low transfection efficacy, higher throughput, and control of transfection dosage and uniformity. Future research needs required to advance micro-/nanofluidics based cell electroporation for broad life science and medical applications are discussed.

Abstract A59: Block/homo polyplex micelle-based GM-CSF gene therapy via intraperitoneal administration elicits antitumor immunity against peritoneal dissemination and exhibits safety potentials in mice and monkeys.

Abstract A block/homo-mixed polyplex micelle, comprising of cationic homo polymer: {N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} P[Asp(DET)] and block copolymer: polyethylene glycol (PEG) -P[Asp(DET)], has been reported to exhibit the efficient transgene expression in vivo by intratracheal and systemic administration. In the present study, we investigated the potential of immunogene therapy by intraperitoneal (i.p.) administration of block/homo polyplex micelles for peritoneal dissemination. For evaluation of transgene expression in vivo, block/homo polyplex micelles showed 12-fold higher level in luciferase expression evaluated by bioluminescence imaging system at 24 h after the i.p. administration compared with block polyplex micelles in nude mice bearing peritoneal dissemination. The distribution of polyplex micelles in tumor nodules was similarly present for both polyplex micelles, where the intracellular uptake of plasmid DNA was detected for block/home polyplex micelles but little for block polyplex micelles. The i.p. administration of block/homo polyplex micelles encapsulating granulocyte macrophage colony stimulating factor (GM-CSF) gene elicited GM-CSF expression dominantly in tumors and less in normal organs (liver and spleen), inhibited the tumor growth and prolonged survival rate for the mice harboring disseminated pancreatic cancer more significantly compared with the mock. The antitumor effect of GM-CSF gene therapy was mediated via the activation of natural killer cells. For safety evaluation, block/homo polyplex micelles indicated almost no adverse events for patho-physical findings and blood examinations in mice and cynomolgus monkeys, although slight increases in serum fibrinogen were observed in the monkey model. In conclusion, block/homo polyplex micelle-based immunogene therapy via i.p. administration may be a safe and effective approach for suppressing intractable peritoneal dissemination. Citation Format: Masahiro Ohgidani, Koichi Furugaki, Shinkai Kentaro, Yumi Kunisawa, Keiji Itaka, Kazunori Kataoka, Kenji Nakano. Block/homo polyplex micelle-based GM-CSF gene therapy via intraperitoneal administration elicits antitumor immunity against peritoneal dissemination and exhibits safety potentials in mice and monkeys. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A59.

Phospholipid micelle encapsulated gadolinium oxide nanoparticles for imaging and gene delivery.

We encapsulated gadolinium oxide (Gd2O3) nanoparticles within phospholipid micelles as a novel low cytotoxic T1-weighted MRI imaging contrast agent (MGdNPs) that can also deliver small molecules such as DNA plasmids. MGdNPs show relatively good MRI relaxivity values, negligible cytotoxicity, excellent cellular uptake and expression of DNA plasmids in vivo. Biodistribution studies in mice show that intranasal and intraperitoneal administration of MGdNPs can effectively target specific organs.

Electroendocytosis Is Driven by the Binding of Electrochemically Produced Protons to the Cell’s Surface

Electroendocytosis involves the exposure of cells to pulsed low electric field and is emerging as a complementary method to electroporation for the incorporation of macromolecules into cells. The present study explores the underlying mechanism of electroendocytosis and its dependence on electrochemical byproducts formed at the electrode interface. Cell suspensions were exposed to pulsed low electric field in a partitioned device where cells are spatially restricted relative to the electrodes. The cellular uptake of dextran-FITC was analyzed by flow cytometery and visualized by confocal microscopy. We first show that uptake occurs only in cells adjacent to the anode. The enhanced uptake near the anode is found to depend on electric current density rather than on electric field strength, in the range of 5 to 65 V/cm. Electrochemically produced oxidative species that impose intracellular oxidative stress, do not play any role in the stimulated uptake. An inverse dependence is found between electrically induced uptake and the solution’s buffer capacity. Electroendocytosis can be mimicked by chemically acidifying the extracellular solution which promotes the enhanced uptake of dextran polymers and the uptake of plasmid DNA. Electrochemical production of protons at the anode interface is responsible for inducing uptake of macromolecules into cells exposed to a pulsed low electric field. Expanding the understanding of the mechanism involved in electric fields induced drug-delivery into cells, is expected to contribute to clinical therapy applications in the future.

Contactless magneto-permeabilization for intracellular plasmid DNA delivery in-vivo

Electroporation, an attractive process for delivering DNA and other molecules into target cells in vivo and in vitro is limited by the necessity of electrodes that need to be in contact with the subject or object to be electroporated. We have used magnetic fields, which do not require material contact with the subject, to temporarily permeabilize cells in guinea pig skin in vivo to enhance uptake and expression of GFP plasmid DNA. The results show for the first time that magnetic fields can trigger a process likely similar to electroporation. In designing the magnetic pulses, our most important criterion was a high rate of change of the magnetic field, based on the principle described by Michael Faraday which is expressed by the formula: E = -dB/dt, (E, electric field, B, magnetic field, t, time). Magnetic fields were generated by a flat electromagnet in a hand-held applicator positioned above the target tissue. The magnetic pulses had a peak magnetic flux density of 4 tesla; 50 pulses were applied in 5 sec. Biphasic magnetic pulses were twice as effective as monophasic pulses and about equally effective as traditional electroporation pulses . Advantages of magnetopermeabilization over electoporation include: No contact between applicator and subject (“contact-less”); no need for invasive, disposable, sterile electrodes (“needle-less”); no pain from needles and reduced overall pain; no known side effects; easier and faster to administer than electroporation; less expensive due to absence of disposables; and, importantly, greater tissue penetration of the magnetic field allowing treatment of anatomical areas inaccessible by electroporation.

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

Cell-surface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer–pDNA transfection

BackgroundInteraction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now. MethodsThe physicochemical properties of the PAMAM–pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay. ResultsPhysicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. ConclusionPresented data shows that the rate-limiting step of PAMAM–pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transgene expression.

Genome-wide screen for Escherichia coli genes involved in repressing cell-to-cell transfer of non-conjugative plasmids

Acquiring new genetic traits by lateral gene transfer is a bacterial strategy for environment adaptation. We previously showed that Escherichia coli could laterally transmit non-conjugative plasmids in co-cultures containing strains with and without the plasmid. In this study, using the Keio collection, a comprehensive library of E. coli knock-out mutants for non-essential genes, we screened for genes responsible for repressing cell-to-cell plasmid transfer in recipient cells. By stepwise screening, we identified 55 ‘transfer-up’ mutants that exhibited approximately 2- to 30-fold increased activities. We confirmed plasmid acquisition by these ‘up’ mutants and revealed that there were no significant changes in antibiotic resistance in the original Keio strains. The presumed functions of these gene products covered a wide range of activities, including metabolism and synthesis, transport, transcription or translation and others. Two competence-gene homologues (ybaV and yhiR) were identified from among these genes. The presumed localizations of these 55 gene products were estimated to be 34 cytoplasmic proteins, 20 in or around the cell surface and 1 unknown location. Our results suggest that these 55 genes may be involved in repressing plasmid uptake during cell-to-cell plasmid transfer.

Function and Regulation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR Associated (Cas) Systems

Phages are the most abundant biological entities on earth and pose a constant challenge to their bacterial hosts. Thus, bacteria have evolved numerous ‘innate’ mechanisms of defense against phage, such as abortive infection or restriction/modification systems. In contrast, the clustered regularly interspaced short palindromic repeats (CRISPR) systems provide acquired, yet heritable, sequence-specific ‘adaptive’ immunity against phage and other horizontally-acquired elements, such as plasmids. Resistance is acquired following viral infection or plasmid uptake when a short sequence of the foreign genome is added to the CRISPR array. CRISPRs are then transcribed and processed, generally by CRISPR associated (Cas) proteins, into short interfering RNAs (crRNAs), which form part of a ribonucleoprotein complex. This complex guides the crRNA to the complementary invading nucleic acid and targets this for degradation. Recently, there have been rapid advances in our understanding of CRISPR/Cas systems. In this review, we will present the current model(s) of the molecular events involved in both the acquisition of immunity and interference stages and will also address recent progress in our knowledge of the regulation of CRISPR/Cas systems.

Oligobenzylethylenimine enriches linear polyethylenimine with a pH-sensitive membrane-disruptive property and leads to enhanced gene delivery activity

We report here the synthesis of a diblock linear polymer of oligo(benzylethylenimine)-b-polyethylenimine (OBzEI-PEI) and investigate its gene delivery properties. The linear copolymer OBzEI-PEI was prepared in a straightforward manner by acidic hydrolysis of a diblock polyoxazoline, which had been made by sequential polymerization of 4-benzyl-2-ethyl-2-oxazoline followed by 2-ethyl-2-oxazoline. pH titration and DNA complexation profiles of the new polymer are similar to regular linear PEIs, but with higher gene transfection efficiencies in various cell lines despite a decreased cellular uptake of plasmid DNA. Further experiments suggest that the OBzEI tail complements the intrinsic proton-sponge endosomolytic activities of PEI with an acid pH-sensitive membrane-perturbing activity.

Development of a Novel Composite Material with Carbon Nanotubes Assisted by Self-Assembled Peptides Designed in Conjunction with β-Sheet Formation

A novel composite material is developed with single-walled carbon nanotubes (SWCNTs) and artificially designed peptides, and its chemical and physicochemical characteristics are evaluated with an aim toward biomedical application. The peptides were designed to form a β-sheet structure that would be suitable for wrapping SWCNTs. The complex of SWCNTs and peptide (SWCNT-peptide) showed good dispersibility in aqueous media and was considerably stable even in the absence of an excess amount of peptide in the media. The formation of SWCNT-peptide was confirmed by its performance in water, atomic force microscopy and transmission electron microscopy observation, and molecular modeling. The possibility of introducing various functions to SWCNT-peptide was also demonstrated by several methods, such as introduction of special amino acids, chemical modification, and additional complex formation based on electrostatic interaction. These results suggest the potential of the SWCNT-peptide complex as a molecular platform on which a desirable structure and/or function can be constructed for biomedical and industrial application.