Carassius auratus-originated recombinant histone H1 C-terminal peptide as gene delivery material

The effective delivery of exogenous genes into eukaryotic cells is important for fundamental and biotechnological research. Protein-based gene delivery including histone proteins has recently emerged as a powerful technique for non-viral DNA transfer. Histones are DNA-binding proteins that function in DNA packaging and protection. In particular, histone H1 is largely responsible for the stabilization of higher-order chromatin structures. Several studies have examined the use of full-length histone H1-mediated gene transfer, and a few studies have investigated the use of C-terminal histone H1 fragments as gene-transfer materials. Previously, we cloned a novel histone H1 cDNA from the goldfish Carassius auratus and found that a recombinant histone H1 C-terminal short peptide (H1C) of 61 amino acids has comparable DNA binding and protection functions as full-length histone H1. In the present work, we successfully expressed and purified soluble recombinant H1C in an Escherichia coli expression system using a hexahistidine tag fusion strategy and providing tRNAs for rare codons. We confirmed its DNA-binding ability and found that this H1C peptide had similar or higher transfection efficiency in mammalian cells (human 293T and mouse NIH/3T3) than the widely used agent lipofectamine. Therefore, we suggest that this novel goldfish-derived recombinant histone H1 C-terminal short peptide could be used as a peptide-based gene-transfer mediator.     

Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA

Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy.     

Efficient transfection of primary zebrafish fibroblasts by nucleofection

Although various gene delivery techniques are available, their application in zebrafish cell cultures has not been extensively studied. Here, we report that nucleofection of zebrafish primary embryonic fibroblasts results in higher transfection efficiency in comparison to other non-viral gene delivery methods. The transfection was performed using green fluorescent protein (GFP) gene constructs of a different size. Greatest DNA uptake was obtained with 4.9-kb plasmid, resulting in 43% GFP positive cells. Nucleofection with 7.4-kb pH2B-GFP plasmid followed by geneticin (G418) selection was successfully used to establish a cell line expressing nuclear histone 2B-GFP fusion protein. Efficient transfection of zebrafish fibroblasts by nucleofection offers a non-viral technique of plasmid delivery and can be used to overexpress genes of interest in these cells.     

Lectin functionalized nanocarriers for gene delivery

Gene therapy has emerged as one of the most promising therapeutic methods to treat various diseases. However, inadequate gene transfection efficacy during gene therapy demands further development of more efficient gene delivery strategies. Targeting genetic material to specific sites of action endows numerous advantages over non-targeted delivery. An ample variety of non-viral gene delivery vectors have been developed in recent years owing to the safety issues raised by viral vectors. Non-viral gene delivery vectors containing specific targeting ligands on their surfaces have been reported to enhance the gene transfection efficiency via receptor-mediated endocytosis for gene delivery. Among various targeting moieties investigated, carbohydrates and lectins (carbohydrate-binding proteins) played an essential role in gene delivery via either direct or reverse lectin targeting strategies. Lectins have a specific carbohydrate binding domain that can bind specifically to the carbohydrates. This review sheds light on various gene delivery nanovectors conjugated with either lectins or carbohydrates for enhanced gene transfection.     

Recombinant mussel adhesive protein as a gene delivery material

Efficient target gene delivery into eukaryotic cells is important for biotechnological research and gene therapy. Gene delivery based on proteins, including histones, has recently emerged as a powerful non-viral DNA transfer technique. Here, we investigated the potential use of a recombinant mussel adhesive protein, hybrid fp-151, as a gene delivery material, in view of its similar basic amino acid composition to histone proteins, and cost-effective and high-level production in Escherichia coli. After confirming DNA binding affinity, we transfected mammalian cells (human 293T and mouse NIH/3T3) with foreign genes using hybrid fp-151 as the gene delivery carrier. Hybrid fp-151 displayed comparable transfection efficiency in both mammalian cell lines, compared to the widely used transfection agent, Lipofectamine 2000. Our results indicate that this mussel adhesive protein may be used as a potential protein-based gene-transfer mediator.     

A recombinant H1 histone-based system for efficient delivery of nucleic acids

We describe here a unique transfer system based on a truncated form of the human linker histone H1F4 for the delivery of nucleic acids to a variety of cells. The efficiency of truncated histone H1.4F was assessed using both primary mammalian and immortalised insect and mammalian cell lines. Our results indicated that recombinant histone H1.4F was able to deliver DNA, dsRNA and siRNA in all cells tested. Quantitative analysis based on reporter gene expression or silencing of target genes revealed that the transfection efficiency of histone H1.4F was comparable to, or better than, liposome-based systems. Notably, the efficiency of histone H1.4F was associated with very low toxicity for transfected cells. The human H1.4F recombinant protein is easily purified in large-scale from bacterial lysates using inexpensive simplified processing. This versatile transfection system represents an important advance in the field of gene delivery and an improvement over earlier nucleic acid delivery methods.     

阳离子聚合物在非病毒基因转染中的研究进展

基因治疗为治疗先天性遗传疾病和严重后天获得性疾病提供了一条新途径.目前,基因载体分为两类:病毒载体和非病毒载体.病毒载体转染效率高,但由于某些病毒载体存在免疫原性、致癌性、宿主DNA插入整合等缺点,从而限制了它们的应用.非病毒载体具有价格低、制备简单、安全有效、无免疫原性等优点,成为基因载体研究的热点.阳离子多聚物是非病毒载体的典型代表.文中综述近年来阳离子多聚物作为基因载体的研究现状和进展,重点介绍了阳离子多聚物基因载体的分类和与DNA的相互作用和传递机制.     

穿膜肽介导的新型基因转运载体的功能研究

目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性,建立-种安全高效、无基因插入片段大小限制的基因转导系统。方法：在TAT-LacI HPM片段C端和N端分别添加GST标签,构建pET-28a（＋）-TAT-LacI HPM-GST和pGEX-GST-TAT-LacI HPM重组表达载体,可溶性表达TAT-LacI HPM-GST及GST-TAT-LacI HPM融合蛋白并纯化,获得TAT-LacI HPM二聚体,免疫荧光检测TAT-LacI HPM融合蛋白穿过HeLa细胞膜的情况,观察EGFP的表达,用免疫印迹检测TAT-LacI HPM融合蛋白介导质粒DNA进入细胞的能力。结果：表达、纯化并获得二聚体融合蛋白,体内实验表明其具有跨膜能力,能介导带有LacI结合序列的DNA质粒进入细胞,并在转染细胞里检测到了目的蛋白。结论：初步证实TAT-LacI HPM融合蛋白作为-种新型通用性非病毒DNA转运载体的可行性,为评价这种新型DNA疫苗载体在提高免疫效果方面的可行性奠定了前期实验基础。     

Enhancement of transfection by physical concentration of DNA at the cell surface

Efficient DNA transfection is critical for biological research and new clinical therapies, but the mechanisms responsible for DNA uptake are unknown. Current nonviral transfection methods, empirically designed to maximize DNA complexation and/or membrane fusion, are amenable to enhancement by a variety of chemicals. These chemicals include particulates, lipids, and polymer complexes that optimize DNA complexation/condensation, membrane fusion, endosomal release, or nuclear targeting, which are the presumed barriers to gene delivery. Most chemical enhancements produce a moderate increase in gene delivery and a limited increase in gene expression. As a result, the efficiency of transfection and level of gene expression after nonviral DNA delivery remain low, suggesting the existence of additional unidentified barriers. Here, we tested the hypothesis that DNA transfection efficiency is limited by a simple physical barrier: low DNA concentration at the cell surface. We used dense silica nanoparticles to concentrate DNA-vector (i.e. DNA-transfection reagent) complexes at the surface of cell monolayers; manipulations that increased complex concentration at the cell surface enhanced transfection efficiency by up to 8.5-fold over the best commercially available transfection reagents. We predict that manipulations aimed at optimizing DNA complexation or membrane fusion have a fundamental physical limit; new methods designed to increase transfection efficiency must increase DNA concentration at the target cell surface without adding to the toxicity.     

Improved gene expression using low molecular weight peptides produced from protamine sulfate

DNA condensation plays a key role in non-viral gene delivery by affecting gene transfection, nuclear targeting, and eventual gene expression efficiency. Theoretically, a DNA condenser with the appropriate DNA condensation ability but without affecting DNA dissociation from DNA condensates inside the cytoplasm should be a perfect carrier for gene delivery. Protamine is a natural DNA condensation agent and has been widely used in gene delivery. In this work, protamine was selectively digested enzymatically to produce low molecular weight protamine fragments (LMWPs) of various lengths and amino acid compositions. The DNA condensation ability and gene transfection efficiency of these LMWP peptides were tested. Compared to protamine, all the LMWP peptides showed lower DNA binding strength. However, some LMWP peptides demonstrated excellent DNA condensation ability and could form very compact DNA condensates with small particle size (∼100 nm). More interestingly, LMWP peptide-mediated in vitro gene delivery showed prolonged (up to 12 days) gene expression. Results from this study suggest that designing DNA condensers with appropriate and tunable DNA binding strengths and condensation abilities would be an effective means to improve gene expression and thus gene therapy efficiency. Since LMWP peptides have low immunogenicity, they would be safer than protamine for use in gene therapies. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 10, pp 1447–1455.     

Intracellular trafficking of polyamidoamine-poly(ethylene glycol) block copolymers in DNA delivery

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have shown immunogenicity and toxicity dangers, but synthetic vectors have lagged in transfection efficiency. Previously, we developed a modular, linear-dendritic block copolymer architecture with high gene transfection efficiency compared to commercial standards. This rationally designed system makes use of a cationic dendritic block to condense the anionic DNA and forms complexes with favorable endosomal escape properties. The linear block provides biocompatibility and protection from serum proteins, and can be functionalized with a targeting ligand. In this work, we quantitate performance of this system with respect to intracellular barriers to gene delivery using both high-throughput and traditional approaches. An image-based, high-throughput assay for endosomal escape is described and applied to the block copolymer system. Nuclear entry is demonstrated to be the most significant barrier to more efficient delivery and will be addressed in future versions of the system.     

Cationic micelle: A promising nanocarrier for gene delivery with high transfection efficiency

Micelles have demonstrated an excellent ability to deliver several different types of therapeutic agents, including chemotherapy drugs, proteins, small‐interfering RNA and DNA, into tumor cells. Cationic micelles, comprising self‐assemblies of amphiphilic cationic polymers, have exhibited tremendous promise with respect to the delivery of therapy genes and gene transfection. To date, research in the field has focused on achieving an enhanced stability of the micellar assembly, prolonged circulation times and controlled release of the gene. This review focuses on the micelles as a nanosized carrier system for gene delivery, the system‐related modifications for cytoplasm release, stability and biocompatibility, and clinic trials. In accordance with the development of synthetic chemistry and self‐assembly technology, the structures and functionalities of micelles can be precisely controlled, and hence the synthetic micelles not only efficiently condense DNA, but also facilitate DNA endocytosis, endosomal escape, DNA uptake and nuclear transport, resulting in a comparable gene transfection of virus.     

Histone H2A significantly enhances in vitro DNA transfection.

BACKGROUND: Gene transfer is a potential treatment modality of genetic disease. Efficient, practical methods of DNA transfection are currently under investigation. MATERIALS AND METHODS: A beta-galactosidase reporter plasmid interacted electrostatically with histones, poly-L-Lys, poly-L-Arg, and a combination of poly-L-Lys and poly-L-Arg. This complex was then used to transfect COS-7 cells. beta-galactosidase activity was quantified and used to compare the efficiency of gene transfection in vitro. A comparison was also made of DNA transfection with the most active histone subclass, i.e., histone H2A, in the absence and presence of an anionic liposome. RESULTS: There was a marked increase in DNA transfection in the presence of histone H2A when compared with the control, whereas each of the other histones and polycations showed little, if any, effect. The extent of activation depends strongly on the DNA/histone ratio and is also a function of the molarity of the final Tris-acetate, pH 8, solution. The anionic liposomes used demonstrated an inhibitory effect. CONCLUSIONS: Histone H2A significantly enhances in vitro DNA transfection whereas other histones and anionic liposomes do not. A study of the difference between histone H2A and other histone subclasses may serve to clarify some of the mechanisms and the essential components of efficient gene delivery.     

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.     

Cross-linked polyethylenimine as potential DNA vector for gene delivery with high efficiency and low cytotoxicity

Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationiccharge potential.High transfection efficiency of PEI,along with its cytotoxicity,strongly depends on itsmolecular weight.To enhance its gene delivery efficiency and minimize cytotoxicity,we have synthesizedsmall cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro.Inthis study,branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate[1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h.The efficiencies of thecross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein(EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines.Flow cytometrywas used to quantify the cellular entry efficiency of plasmid and the transgene expression level.Thecytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay.EGDMA-PEI 800-4h,atypical cross-linked PEI reported here,mediated a more efficient expression of reporter gene than thecommercially available 25-kDa branched PEI control,and resulted in a 9-fold increase in gene deliveryin B16F10 cells and a 16-fold increase in 293T cells,while no cytotoxicity was found at the optimizedcondition for gene delivery.Furthermore,the transfection activity of polyplexes was preserved in thepresence of serum proteins.     

Nature as a source of inspiration for cationic lipid synthesis

Synthetic gene delivery systems represent an attractive alternative to viral vectors for DNA transfection. Cationic lipids are one of the most widely used non-viral vectors for the delivery of DNA into cultured cells and are easily synthesized, leading to a large variety of well-characterized molecules. This review discusses strategies for the design of efficient cationic lipids that overcome the critical barriers of in vitro transfection. A particular focus is placed on natural hydrophilic headgroups and lipophilic tails that have been used to synthesize biocompatible and non-toxic cationic lipids. We also present chemical features that have been investigated to enhance the transfection efficiency of cationic lipids by promoting the escape of lipoplexes from the endosomal compartment and DNA release from DNA-liposome complexes. Transfection efficiency studies using these strategies are likely to improve the understanding of the mechanism of cationic lipid-mediated gene delivery and to help the rational design of novel cationic lipids.     

Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation

The goals of this study were to identify mammalian cell lines which could be efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS-7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporation for DNA delivery and green fluorescent protein (Evans, 1996) as the reporter gene. In addition, we have evaluated the effects of stable expression of viral proteins, cell cycle manipulation, and butyrate post-treatment in small scale experiments. The cell lines varied widely in their GFP transfection efficiencies. Stable expression of simian virus 40 large T-antigen or Epstein Barr nuclear antigen failed to significantly increase transfection efficiency above that seen in the parental lines. Aphidicolin (a DNA polymerase inhibitor), which blocked cells from S or G2/M, brought about an increase in transfection efficiency in two cell lines. The primary effect of butyrate (a histone deacetylase inhibitor) post-treatment was an increased intensity of the fluorescent signal of green fluorescent protein, as measured by flow cytometry (1.0 to 4.2-fold, depending on the cell line). The combined use of aphidicolin pretreatment followed by butyrate treatment post- electroporation yielded increases in fluorescence intensities ranging from 0.9 to 6.8-fold. Based on their high transfection efficiencies in small scale experiments, rapid growth, and ability to grow in suspension culture, CHO, CHO-TAg, and 293-EBNA were selected to assess the feasibility of using flow electroporation for large-scale transfections. Using secreted placental alkaline phosphatase as a reporter, 293-EBNA cells produced the highest protein levels in both the presence and absence of butyrate. These data indicate that flow electroporation provides an efficient method of DNA delivery into large numbers of cells for mammalian protein production. This revised version was published online in August 2006 with corrections to the Cover Date.     

Substrate-mediated nucleic acid delivery from self-assembled monolayers

Substrate-mediated nucleic acid (NA) delivery involves the immobilization of NAs or NA delivery vehicles to biomaterials for localized transfection of cells. Self-assembled monolayers (SAMs) offer an easy system to immobilize delivery vectors. SAMs form well-defined surfaces; therefore, the effect of surface composition on vector immobilization and transfection efficiency can also be studied. To date, the most effective SAM-mediated delivery systems have utilized nonspecific interactions for immobilization; however, systems that rely on specific interactions between vector and surface can impart higher control of spatial and/or temporal delivery. This review summarizes systems that use both specific and nonspecific interactions for gene delivery from SAMs; highlights progress and remaining challenges; and explores other specific recognition modalities that might be employed for future applications in surface-mediated NA delivery.     

PEG修饰的聚阳离子基因载体PEG-Polycarbam-SP在COS-7细胞中转染和毒性的研究

目的：寻找一种转染效率高,细胞毒性低的非病毒基因载体,研究以人体内源性精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以聚乙二醇（PEG）作为亲水基团连接剂合成亲水修饰聚阳离子载体PEG-Polycarbam-SP的基因担载效率,以及对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：琼脂糖凝胶电泳方法考察复合物的基因担载效率,检测基因复合物的粒径和电位,以荧光素酶质粒为报告基因,研究PEG-Polycarbam-SP/DNA的复合物在COS-7细胞的转染活性,用MTT方法研究PEG-Polycarbam-SP对COS-7细胞的毒性。结果：聚合物与质粒在质量比5以后形成的复合物粒径稳定在50nm左右,Zate电位在20mV左右。COS-7细胞实验显示PEG-Polycarbam-SP具有低于PEI 25kDa的细胞毒性,同时也具有高效输送DNA的能力。结论：PEG-Polycarbam-SP是一种新型的高效、低毒,在基因治疗领域有潜在应用价值的非病毒基因输送载体。     

Development of a novel fusogenic viral liposome system (HVJ-liposomes) and its applications to the treatment of acquired diseases

Toward human gene therapy and gene analysis in vivo, a novel hybrid vector based on liposome has been developed for more efficient gene delivery and gene expression. The liposome was decorated with HVJ (Sendai virus) envelope fusion proteins to introduce DNA directly into the cytoplasm, and contained DNA and DNA-binding nucelar protein to enhance expression of the gene. Recently, several types of HVJ-liposomes were developed by altering the lipid components of the liposomes. HVJ-cationic liposomes increased gene delivery 100 - 800 times more efficiently in vitro than the conventional HVJ-anionic liposomes. HVJ-cationic liposomes were also more useful for gene expression in restricted portions of organs and for gene therapy of disseminated cancers. It was further discovered that the use of anionic liposomes with a virus-mimicking lipid composition (HVJ-AVE liposomes) increased transfection efficiency by several fold in vivo, especially in liver and muscle. By coupling the Epstein-Barr (EB) virus replicon apparatus to HVJ-liposomes, transgene expression was sustained in vitro and in vivo. Most animal organs were found to be suitable targets for the fusigenicviral liposome system, and numerous gene therapy strategies using this system were successful in animals.