Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60–80% to 30–40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.     

Layered double hydroxide nanoparticles as cellular delivery vectors of supercoiled plasmid DNA

We prepared stable homogeneous suspensions with layered double hydroxide (LDH) nanoparticles for in vitro gene delivery tests. The viability of HEK 293T cells in the presence of LDH nanoparticles at different concentrations was investigated. This revealed 50% cell viability at 500 microg/mL of LDH nanoparticles that is much higher than 50-100 microg/mL used for the delivery tests. The supercoiled pEF-eGFP plasmid (ca. 6100 base pairs) was mixed with LDH nanoparticle suspensions for anion exchange at a weight ratio of DNA/LDH between 1:25 and 1:100. In vitro experiments show that GFP expression in HEK 293T cells starts in the first day, reaches the maximum levels by the second day and continues in the third day. The GFP expression generally increases with the increase in DNA loading in DNA-LDH nanohybrids. However, the delivery efficiency with LDH nanoparticles as the agent is low. For example, the relative efficiency is 7%-15% of that of the commercial agent FuGENE 6. Three to 6% of total cells expressed GFP in an amount detectable by the FACS cytometry 2 days after transfection at 1 microg/mL of plasmid DNA with 25 microg/mL of LDH nanomaterial. The lower delivery efficiency could be attributed to the aggregation of LDH nanoparticles caused by the long-chain plasmid DNA.     

Two-wavelength fluorescence assay for DNA repair

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP). Both proteins can be excited by visible light with a maximum at 488 nm, but EGFP emits with a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This makes it possible to monitor the expression of the two genes simultaneously by measuring the fluorescence at two wavelengths. HEK293 cells were cotransfected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At different time intervals after transfection the fluorescence of EGFP was determined relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used to calculate the number of UV lesions in DNA and hence the repair capacity of the host cells. It was found that HEK293 cells were able to repair approximately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average.     

Transfection of partially purified plasmid DNA for high level transient protein expression in HEK293-EBNA cells

One of the major constraints to performing large-scale transfections of cultured mammalian cells for the transient expression of recombinant proteins is the production of large quantities of purified plasmid DNA. In this report partially purified plasmid DNA was prepared by a method that combines alkaline lysis of E. coli with standard precipitation techniques. The efficiency of calcium phosphate-DNA co-precipitate formation with crude DNA was similar to that observed for pure DNA, but precipitate formed with crude DNA also contained RNA. The transfection of adherent and suspension-adapted HEK293-EBNA cells with partially purified pEGFPN1 resulted in levels of transient GFP expression equivalent to those achieved with pure DNA. In addition, the co-transfection of 1-200 ml cultures of suspension-adapted HEK293-EBNA cells with two different plasmids encoding the heavy and light chain genes of anti-human RhD IgG1, respectively, yielded similar IgG titers with pure and partially purified plasmid DNA. Finally, it was observed that suspension-adapted cells were more tolerant to the presence of RNA in the plasmid preparations than were adherent cells. These findings are relevant to the field of DNA transfection, including applications ranging from high-throughput screening to large-scale transient protein expression.     

Amphiphilic peptides with arginines and valines for the delivery of plasmid DNA

A non-toxic and efficient gene carrier is one requirement for clinical gene therapy. In this study, amphiphilic peptides composed of arginines and valines were synthesized and characterized as plasmid DNA (pDNA) carriers. The peptides have a cationic region containing 1-4 arginines and a hydrophobic region containing 6 valines. The arginine-valine peptides (RV peptides) formed micelles in aqueous solution with a critical micelle concentration (CMC) of 1.35 mg/ml. In gel retardation assay, the RV peptides retarded all pDNA at weight ratios (pDNA:RV peptide) of 1:3 for R1V6, 1:2 for R2V6 and R3V6, and 1:1 for R4V6. A heparin competition assay showed that the R3V6 peptide formed tighter complexes with pDNA than poly-L-lysine (PLL). In vitro transfection assay into HEK293 cells showed that the R1V6 and R2V6 peptides had the highest transfection efficiencies at 1:30 weight ratios (pDNA:RV peptide), while the R3V6 and R4V6 peptides had the highest efficiencies at 1:20 weight ratios. Under optimal conditions, the R3V6 peptide had the highest transfection efficiency of all the RV peptides and PLL. MTT assay showed that the RV peptides did not have any detectable toxicity to cells. Therefore, the RV peptide may be useful for the development of non-toxic gene carriers.     

Transient gene expression in mammalian cells grown in serum-free suspension culture

In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

An archaeal histone-like protein as an efficient DNA carrier in gene transfer

HPhA, a recombinant histone-like protein from Pyrococcus horikoshii OT3 strain, has compacting activity with DNA as previously reported. The extreme stability and DNA packaging activity of the HPhA make it a candidate as a DNA carrier. Here, the plasmid DNA-HPhA complexes were fully characterized by gel retardation assay and DNase resistance assay. It was further proved that HPhA has in vitro DNA transfection activity. HPhA-mediated transfection efficiency was dependent on the mass ratio of HPhA to DNA, the incubation time and the presence of calcium. A protocol for HPhA-mediated transfection in vitro was established to improve transfection efficiency. The optimal mass ratio of HPhA to DNA was 6:1, and the incubation time required for the DNA-HPhA complex to be in contact with the cell was 4 h. In addition, the presence of 2 mM CaCl2 in the cell culture medium was required for efficient transfection. Serum did not show inhibition of HPhA-mediated transfection. Most importantly, the cytotoxicity of HPhA is lower than that of commonly used cationic liposome-based gene delivery systems, and HPhA-mediated transfection in NIH 3T3, HEK 293, HL-7702, HepG2 and Cos 7 cell lines in vitro has a higher efficiency and reproducibility. These results demonstrate that the HPhA is a new, potentially widely applicable and highly efficient gene carrier.     

人P2X7真核表达载体的构建及稳定转染细胞株的建立

目的：构建人P2X7基因的真核表达载体,并通过转染获得稳定表达P2X7分子的HEK293细胞株。方法：以人脑组织P2X7cDNA为模板扩增出P2X7基因,插入到真核表达载体pEGFP-N1中,构建重组质粒pEGFP-N1/P2X7。用X-fect试剂盒将重组质粒转染HEK293细胞,通过G418辅助荧光筛选建立稳定表达P2X7-EGFP细胞株。经流式细胞仪、Western blot和激光共聚焦显微镜检测,了解人P2X7在HEK293细胞中的表达水平及细胞内定位。结果：重组质粒pEGFP-N1/P2X7构建正确,建立了稳定表达人P2X7的HEK293细胞系。Western blot和流式细胞仪检测证实,P2X7在HEK293细胞系中成功表达,激光共聚焦显微镜检测显示P2X7-EGFP定位在细胞膜上。结论：重组载体pEGFP-N1/P2X7构建成功并建立了稳定表达人P2X7的HEK293细胞系,为进一步研究P2X7离子通道结构和功能奠定基础。     

G418抗性HEK293细胞的培育

目的　培育具有G418抗性的HEK2 93细胞 ,用于建立猪内源性反转录病毒感染人HEK2 93细胞的模型。方法　通过脂质体转染的方法 ,将含有neo基因的质粒pIRESneo导入HEK2 93细胞中 ,利用G418的选择特性 ,对转染细胞进行压力筛选 ,并对其进行了PCR鉴定。结果　经 6 0 0 μg ml的G418压力筛选后 ,获得了抗性细胞克隆。抗性细胞的形态和生长速度与筛选前细胞没有差异 ,特异性核苷酸引物检测抗性细胞基因组DNA ,可以扩增出对应的核苷酸片段。结论　成功地培育了G418抗性HEK2 93细胞 ,为建立猪内源性反转录病毒感染人HEK2 93细胞的模型奠定了基础。     

Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu mediate efficient non-viral gene delivery

BACKGROUND: The inherent ability of certain peptides or proteins of viral, prokaryotic and eukaryotic origin to bind DNA was used to generate novel peptide-based DNA delivery protocols. We have developed a recombinant approach to make fusion proteins with motifs for DNA-binding ability, Mu and membrane transduction domains, TAT, and tested them for their DNA-binding, uptake and transfection efficiencies. In one of the constructs, the recombinant plasmid was designed to encode the Mu moiety of sequence MRRAHHRRRRASHRRMRGG in-frame with TAT of sequence YGRKKRRQRRR to generate TAT-Mu, while the other two constructs, Mu and Mu-Mu, harbor a single copy or two copies of the Mu moiety. METHODS: Recombinant his-tag fusion proteins TAT-Mu, Mu and Mu-Mu were purified by overexpression of plasmid constructs using cobalt-based affinity resins. The peptides were characterized for their size and interaction with DNA, complexed with plasmid pCMVbeta-gal, and shown to transfect MCF-7, COS and CHOK-1 cells efficiently. RESULTS: Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu were cloned and overexpressed in BL21(DE3)pLysS with greater than 95% purity. The molecular weight of TAT-Mu was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to be 11.34 kDa while those of Mu and Mu-Mu were 7.78 and 9.83 kDa, respectively. Live uptake analysis of TAT-Mu, Mu and Mu-Mu as DP (DNA+peptide) or DPL (DNA+peptide+lipid) complexes into MCF-7 cells, followed by immunostaining and laser scanning confocal microscopy, demonstrated that the complexes are internalized very efficiently and localized in the nucleus. DNA:peptide complexes (DP) transfect MCF-7, COS and CHOK-1 cells. The addition of cationic liposomes enhances the uptake of the ternary complexes (DPL) further and also brings about 3-7-fold enhancement in reporter gene expression compared to DP alone. CONCLUSIONS: Recombinant proteins that are heterologous fusions, having DNA-binding domains and nuclear localization epitopes, generated in this study have considerable potential to facilitate DNA delivery and enhance transfection. The domains in these fusion proteins would be promising in the development of non-viral gene delivery vectors particularly in cells that do not divide.     

Delivery and pathway in MCF7 cells of DNA vectorized by cationic liposomes derived from cholesterol

We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.     

Hyperbranched poly(amino ester)s with different terminal amine groups for DNA delivery

Hyperbranched poly(amino ester)s containing tertiary amines in the core and primary, secondary, and tertiary amines in the periphery, respectively, were evaluated for DNA delivery in vitro. The same core structure facilitated the investigation on the effects of the terminal amine type on the properties of hyperbranched poly(amino ester)s for DNA delivery. The hydrolysis of the poly(amino ester)s was monitored using (1)H NMR. The results reflected that the terminal amine type had negligible effects on the hydrolysis rate but was much slower than that of linear poly(amino ester)s, probably due to the compact hyperbranched spatial structure preventing the accessibility of water. In comparison with PEI 25 K, the hyperbranched poly(amino ester)s showed much lower cytotoxicity in Cos7, HEK293, and HepG2 cells. Gel electrophoresis indicated that poly(amino ester)s could condense DNA efficiently, and the zeta potentials and sizes of the complexes formed with different weight ratios of hyperbranched poly(amino ester)s and DNA were measured. Remarkably, all the hyperbranched poly(amino ester)s showed DNA transfection efficiency comparable to PEI 25 K in Cos7, HEK293, and HepG2 cells regardless of the terminal amine type. Therefore, the terminal amine type had insignificant effects on the hydrolysis rate, cytotoxicity, DNA condensation capability, and in vitro DNA transfection efficiency of the hyperbranched poly(amino ester)s.     

Use of a quaternary ammonium detergent in liposome mediated DNA transfection of mouse L-cells

Sonicated liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and a quaternary ammonium detergent (dodecyl-, tetradecyl-, or cetyl-trimethylammonium bromide) mediates functional transfer of pSV2 CAT plasmid DNA to mouse L929 fibroblasts. Successful transfection was determined by assaying for chloramphenicol acetyltransferase activity in cell lysates collected 40 h after exposure to the lipid-DNA complexes. Liposomes prepared with the quaternary ammonium detergents were less toxic than the free detergents at the same concentrations and were more efficient in their delivery of the plasmid DNA to the cells. Analysis of the three detergents in combination with the lipid showed that cetyltrimethylammonium bromide was least toxic to the cells. This detergent, at a minimal concentration of 20 mol% in DOPE, allowed for stable liposome preparations and efficient transfection. Optimal efficiency of transfection occurred with 30 micrograms of DNA. Further increases in the DNA concentration caused a decrease in the transfection efficiency, perhaps due to charge repulsions between the liposomes now saturated with negatively charged DNA and the negatively charged cell surface. The transfection activity of the liposome was limited by its cytotoxicity at high liposome concentrations. These results are compared with that of the Lipofectin, another positively charged liposome preparation which is commercially available. Although the overall transfection activity of the liposome containing the quaternary ammonium detergent is somewhat lower than that of the Lipofectin, it may serve as an inexpensive and convenient alternative.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis

The present study investigated whether protamine sulfate can be used to improve the efficiency of bovine sperm mediated transgenesis (SMT) by protecting the plasmid pCX-EGFP against nuclease activity. A high proportion (31%) of bovine spermatozoa transfected with the plasmid pCX-EGFP maintain their motility. Using an in vitro assay, protamine sulfate protected the plasmid against degradation by DNase I. However, upon transfecting spermatozoa, the plasmid remained intact regardless of whether it was complexed to protamine sulfate. When in vitro fertilisation (IVF) was undertaken using transfected sperm, 14.6 and 10.2% of blastocysts derived from pCX-EGFP only and pCX-EGFP-protamine transfected sperm, respectively, were PCR positive for the plasmid. In conclusion, using spermatozoa transfected with either pCX-EGFP or pCX-EGFP-protamine complexes, produced PCR positive blastocysts after SMT. However, the use of protamine sulfate does not improve the efficiency of SMT suggesting that factors other than nuclease activity could be limiting.     

Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis

The green fluorescent protein (GFP) has attracted much interest as a reporter for gene expression. In this paper, application of capillary electrophoresis with laser-induced fluorescent (CE-LIF) for quantitation of green fluorescence protein in cellular extracts and single cells is investigated. The S65T mutant form of GFP protein was successfully expressed in human embryonic kidney (HEK293) cells, and its production was confirmed by fluorescence microscopy and CE-LIF. The mass limit of detection for the mutant S65T was 5.3 x 10(-20) mol, which was better than that for the wild-type GFP by a factor of six. Detection of a small amount of GFP is difficult by conventional techniques such as fluorescent microscopy due to interference from cell autofluorescence at low GFP concentrations. The HEK293 cells were transfected with the GFP plasmid that produced S65T-GFP. Transient production of S65T protein was detected 2 h after the transfection and reached a maximum after 48 h. The protein concentration began to decrease significantly after 96 h. Single cell analysis of HEK293 cells after transfection with GFP plasmid indicate a nonuniform production of S65T-GFP protein among cells.     

Gold nanoparticle-mediated transfection of mammalian cells.

Mixed monolayer protected gold clusters (MMPCs) functionalized with quaternary ammonium chains efficiently transfect mammalian cell cultures, as determined through beta-galactosidase transfer and activity. The success of these transfection assemblies depended on several variables, including the ratio of DNA to nanoparticle during the incubation period, the number of charged substituents in the monolayer core, and the hydrophobic packing surrounding these amines. Complexes of MMPCs and plasmid DNA formed at w/w ratios of 30 were most effective in promoting transfection of 293T cells in the presence of 10% serum and 100 microM chloroquine. The most efficient nanoparticle studied (MMPC 7) was approximately 8-fold more effective than 60 kDa polyethylenimine, a widely used transfection agent.     

Enhancement of transfection efficiency by protamine in DDAB lipid vesicle-mediated gene transfer.

We have previously developed a simple gene transfection procedure mediated by cationic lipid vesicles for animal cells, in which a commercially available cationic surfactant, dimethyldioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. In the present study, we examined enhancement of transfection efficiency for this method by adding protamine to plasmid DNA solution before the formation of DNA/lipid vesicle complexes. Both free-base protamine and protamine sulfate provided enhanced transfection efficiency and expression level, but the optimal amount of the two protamines was different. The enhancement in transfection efficiency and expression level by protamines was observed in all the cell lines (COS-7, Hela, NIH3T3, MDCK, and BHK-21C13) and all the plasmids (pCMVbeta, pmiwZ, and pCH110) tested. The enhancement in both transfection efficiency and expression level was at most 20-fold compared with that using only DDAB lipid vesicles. Protamines seemed to protect DNA from degradation by DNase and promote DNA delivery into a nucleus.     

Co-transfection and tandem transfection of HEK293A cells for overexpression and RNAi experiments

pIRES2-EGFP was employed and a non-target shRNA expressing plasmid was constructed to simulate overexpression and RNAi (RNA interference) experiments. Transfection of pIRES2-EGFP into HEK293A cells by cationic lipids VigoFect demonstrated that transfection efficiency increased in a dose-dependent manner with amount of DNA plasmid used, and optimal transfection time and cell density should be identified to reach a compromise of higher transfection efficiency and lower toxicity. Co-transfection experiments indicated that the two co-transfected plasmids were equivalently delivered into the same cells, and the co-transfection efficiency was rarely affected by cell density and proportion of the two plasmids. However, plasmid-receipted cells seemed indisposed to accept plasmid again during the second transfection, and very low co-transfection efficiency was observed in tandem transfection.     

Electrodissolution of inorganic ions/DNA multilayer film for tunable DNA release

A layer-by-layer film composed of DNA and inorganic zirconium ion (Zr(4+)) was fabricated on the surface of gold thin film, and an electric field triggered disintegration of the multilayer film was studied by using electrochemical surface plasmon resonance (EC-SPR). EC-SPR results demonstrated that the film was disassembled upon the application of an electric field and the disassembly rate varied with the applied potential, leading to the controlled release of DNA. The electrodissolution could be switched off by removing the electric potential and reactivated by reapplying the potential. By incorporating plasmid DNA (pDNA) in to this controlled release system, the multilayer film could sustain the consecutive release of pDNA electrochemically. The released pDNA retained its integrity and transfection activity, and expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. The electrochemical systems, with advantages of miniaturization, surface-tailoring, safety, simplicity, convenience, automation, low-cost, and free of immune reactions, made the electrical route a very attractive gene-delivery alternative.