Cationic Liposomes Containing Disulfide Bonds in Delivery of Plasmid DNA

Abstract

Cationic liposomes are non-viral gene transfer vectors for in vitro and in vivo experiments. In the present studies, we investigated whether a disulfide linkage in a cationic lipid was reducible by cell lysate resulting in the release of plasmid DNA and enhanced gene transfection. We also investigated if the differences in transgene production were from differences in total amount of cellular associated plasmid DNA. We systematically compared the gene transfection of disulfide bond containing-cationic lipid, 1', 2'-dioleoyl-sn-glycero-3'-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO), its non-disulfide-containing analog, 1', 2'-dioleyl-sn-glycero-3'-succinyl-1, 6-hexanediol ornithine conjugate (DOGSHDO), 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP). Two transgene reporter systems (i.e., luciferase and green fluorescent protein (GFP)) were used to address transgene transgene expression and transgene efficiency. Experiments with the luciferase expression plasmid resulted in transgene activity up to 11 times greater transgene production for the disulfide containing lipid in at least two different cell lines, COS 1 and CHO cells. When transgene expression was determined by GFP activity, DOGSDSO liposomes were four times greater than the non-disulfide lipid or positive control (DOTAP) liposomes. By quantifying nucleic acid uptake by flow cytometry it was also demonstrated that increase expression was not solely from an increase in cellular plasmid DNA accumulation. These results demonstrate that cationic lipids containing a disulfide linkage are a promising method for gene transfer.     

结核分枝杆菌Rv3194c蛋白的亚细胞定位

【目的】Rv3194c基因编码的是结核分枝杆菌的PDZ信号蛋白,本研究探讨该蛋白的亚细胞定位,为其细胞结合蛋白的筛选奠定基础。【方法】从H37Rv基因组中扩增出编码只含有PDZ结构域的tRv3194c (Rv3194c 1–234 aa)的基因片段,在3′端加T2A和EGFP序列,一并插入真核表达载体构建出pcDNA3.1-tRv3194c-T2A-EGFP。将构建好的质粒瞬时转染L929细胞,并共感染重组痘苗病毒vTF7-3,用间接免疫荧光、流式细胞分选以及Western blotting检测融合蛋白的表达以及亚细胞定位。【结果】成功构建出真核表达载体pcDNA3.1-tRv3194c-T2A-EGFP,瞬时转染L929细胞后融合蛋白tRv3194c定位于线粒体膜上,且重组痘苗病毒vTF7-3的感染有助于靶蛋白表达水平的提高。【结论】Rv3194蛋白的PDZ结构域与线粒体外膜相关蛋白结合,为了解该蛋白在细胞内的致病机制提供重要线索。     

Cholesterol depletion induced by RNA interference targeting DHCR24 protects cells from liposome-induced cytotoxicity

Abstract

Existing evidence has demonstrated liposomes as the gene transporter induce the cytotoxicity during the transfection process through several known pathways. In the present study, we investigated the possibility of siRNAs targeting 3-β-hydroxysterol △-24-reductase (DHCR24), which encodes an enzyme catalyzing the last step of cholesterol biosynthesis, to suppress the liposome cytotoxicity induced by lipid-based transfection reagent in the neuroblastoma cell line N2A. We found that the siRNAs targeting DHCR24 mRNA protect cells from the liposome-induced cell death, probably through the effect of siDHCR24s on the reduction of the cellular cholesterol and decrease in the generation of reactive oxygen species (ROS). This suggests that siRNAs targeting DHCR24 or other methods that reduce the intracellular cholesterol levels might be a good strategy for avoiding the cytotoxicity of liposomes, without impairing its efficiency of gene-delivering.     

Antisense Oligodeoxynucleotide Complementary to CXCR4 mRNA Block Replication of HIV-1 in COS Cells

Abstract

CXCR4 is both a chemokine receptor and an entry co-receptor for the T-cell line-adapted human immunodeficiency virus type 1 (HIV-1). To find a more efficacious therapeutic treatement of acquied immunodeficiency syndrome, we exmined the effects of antisense oligonucleotides on CXCR4 production. COS cells, stably expressing CXCR4 and CD4, were incubated with several kinds of oligonucleotides. Total human p24 antigen production was determined using an enzyme-linked immunosorbent assay system. An antisense phosphorothioate-modified oligonucleotide, complementary to the translation region of the CXCR4 mRNA, showed minimal inhibition of p24 antigen production at the high concentration of 2μM. On the other hand, the antisense phosphorothioate oligonucleotide, when used with transfection reagents, showed high efficiency at low concentrations, and confirmed the sequence-specific action. Interestingly, the oligonucleotide with the natual phosphodiester backbone, when used with the transfection reagents, also had high functional effects, comparable to the modified oligonucleotide. This defines the prerequisite criteria necessary for the design and the application of antisense oligonucleotides against HIV-1 in vivo.     

High-frequency Protoplast-transfection of Streptomyces parvulus 2297 with Actinophage R4 DNA

High frequency transfection of Streptomyces parvulus with actinophage R4 DNA was performed by modifying the procedure of protoplast transformation of S. coelicolor A3(2) with SCP2 plasmid DNA [Bibb et al., Nature, 274, 398 (1978)]. Optimum conditions for protoplast transfection included the presence of 16~24% (w/v) polyethyleneglycol 4000, and the maximum efficiency of transfection was 3 × 10^?5 per phage DNA molecule. This value was at least 100 times higher than the efficiency of previously reported transfection systems in Streptomyces.     

Transiently redirected T cells for adoptive transfer

Background aimsT cells can be redirected to reject cancer by retroviral transduction with a chimeric antigen receptor (CAR) or by administration of a bispecific T cell engager (BiTE). We demonstrate that transfection of T cells with messenger (m) RNA coding for CAR is an alternative strategy.MethodsWe describe the pre-clinical evaluation of a method based on transient modification of expanded T cells with a CD19 CAR directed against B-cell malignancies. CAR mRNA was generated under cell-free conditions in a scalable process using recombinant RNA polymerase. Efficient and non-toxic square-wave electroporation was used to load the mRNA into the cytoplasm of T cells with no risk of insertional mutagenesis.ResultsAfter transfection > 80% of T cells were viable, with 94% CAR expression. Transfected T cells were cytolytic to CD19⁺ targets and produced interferon (IFN)-γ in response. Killing of CD19⁺ target cells was demonstrated even at day 8 with undetectable CAR expression. Increasing the concentration of mRNA resulted in higher surface CAR expression, better killing and more IFN-γ release but at the expense of increased activation-induced cell death. Finally, we demonstrated that a second transgene could be introduced by co-electroporation of CXCR4 or CCR7 with CAR to also modify chemotactic responses.ConclusionsWe advocate the transient redirection approach as well suited to meet safety aspects for early phase studies, prior to trials using stably transduced cells once CAR has been proven safe. The simplicity of this methodology also facilitates rapid screening of candidate targets and novel receptors in pre-clinical studies.     

Avoiding untoward immune responses to novel gene products

For successful therapeutic transfection of a missing (or corrective) gene, by the use of liposomes or other delivery systems, it is essential that the patient be immunosuppressed to the corresponding immunogenic, transgenic protein prior to its transfection. We have developed a method for antigen-specific, long-term suppression of de novo induction of both antibodies and cytotoxic T lymphocytes--in spite of repeated administration of the antigen in question over extended periods. The method consists of converting the antigen to its tolerogenic derivative by coupling it to an appropriate number of molecules of monomethoxypolyethylene glycol (mPEG).     

Inclusion of a single-tail amino acid-based amphiphile in a lipoplex formulation: Effects on transfection efficiency and physicochemical properties

Abstract

Effects of the addition of a cationic amino acid-based synthetic amphiphile, arginine N-lauroyl amide dihydrochloride (ALA), to a lipid-based transfection formulation have been investigated. It is shown that the inclusion of ALA results in a substantial enhancement of the transfection capability of lipoplexes prepared with liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine and cholesterol, which themselves mediate highly efficient transfection. A possible explanation for the increased biological activity is that ALA adsorbed to the surface of the DNA-lipid complexes is involved in triggering internalization. However, in order to identify possible additional factors underlying the enhanced transfection efficiency, the physical properties of formulations with and without ALA were characterized using cryo-transmission electron microscopy, dynamic light scattering, and an ethidium bromide intercalation assay. ALA seems to have limited influence on the initial internal structure of the complexes and the protection of DNA, but its presence is found to decrease the average effective size of the dispersed particles; this change in size may be important in improving the biological activity. Furthermore, ALA can act to influence the transfection efficiency of the formulation by promoting the release of DNA following internalization in the transfected cells.     

A Radioimmunoassay-Based Method for Measuring the True Affinity of a Monoclonal Antibody with Trace Amounts of Radioactive Antigen: Illustration with the Products of a Cell-Free Protein Synthesis System

This communication describes a novel highly sensitive method for measuring the affinity of a monoclonal antibody for its antigen. It is based on a radioimmunoassay in which the antigen is labeled with radioactivity. It is therefore particularly well adapted to the study of trace amounts of radiolabeled polypeptide chains produced either in vivo, or in vitro by a cell free protein synthesis system or by chemical radiolabeling. It offers several advantages over previously described methods. Though making use of insolubilized antibody, it does measure the true affinity constant of the monoclonal antibody in solution for the antigen. It can be used even when the antigen is present at concentrations far below the dissociation constant of the antibody/antigen complex. It does not require the antigen or the antibody to be purified. In most cases, it requires no sophisticated equipment. This method could be easily adapted to the determination of the equilibrium constant of any type of protein/ligand system.     

Synthesis and characterization of some metal complexes of a proton transfer salt,and their inhibition studies on carbonic anhydrase isozymes and the evaluation of the results by statistical analysis

Abstract

A novel proton transfer compound (HMeOABT)?⁺?(HDPC)^? (1) and its Fe(III), Co(II), Ni(II) and Cu(II) complexes (2–5) have been prepared and characterized by spectroscopic techniques. Complex 4 has distorted octahedral conformation revealed by single crystal X-ray diffraction method. Structures of the other complexes might be proposed as octahedral according to experimental data. All compounds were also evaluated for their in vitro inhibition effects on hCA I and II for their hydratase and esterase activities. Although there is no inhibition for hydratase activities, all compounds have inhibited the esterase activities of hCA I and II. Data have been analyzed by using a one-way analysis of variance. The comparison of the inhibition studies of 1–5 to parent compounds indicates that 1–5 have superior inhibitory effects. The inhibition effects of 2–5 are also compared to inhibitory properties of the metal complexes of MeOABT and H2DPC, revealing an improved transfection profile.     

Recombination in bacteriophage P2: recA dependent enhancement by ultraviolet irradiation and by transfection with mixed DNA dimers

Summary Bacteriophage P2 is known for its exceptionally low rate of spontaneous (non-integrative) recombination, which however may be stimulated by ultraviolet irradiation of the phage. We show here that ligated dimers, made in vitro from mixtures of DNAs of two P2 mutants, upon transfection of lysozyme-spheroplasts give origin to recombinants at high frequency. While spontaneous P2 recombination occurs independently of the main recombination pathway of the bacteria, P2 recombinant formation following either ultraviolet irradiation or transfection with DNA dimers requires at least some element of such a pathway, since it is absent or greatly reduced in recA ^- bacteria or spheroplasts. It would seem that, in the course of its lytic development, P2 deploys a mechanism that inhibits the main recombination pathway of the host cell, or assumes DNA configurations refractory to it.     

PEN-2 Overexpression Induces γ-Secretase Protein and its Activity with Amyloid β-42 Production

PEN-2 is a component of the γ-secretase complex, which is involved in the cleavage of the β-amyloid precursor protein. The aim of this study was to determine the mechanism by which PEN-2 overexpression regulates γ-secretase expression and the production of Aβ-42. In order to determine this, a hybrid gene harboring human PEN-2 was constructed, and used in the transfection of SK-N-MC human neuroepitheliomal cells. This cell line was also co-transfected with a combination of human mutant presenilin 2 (hPS2m) and APPsw. Our results indicated that (i) human PEN-2 overexpression induced an increase in γ-secretase activity and its proteins, including PS1-CTF, APH-1, and nicastrin, thus production of Aβ-42, (ii) co-transfection of human PEN-2 with both hPS2m and APPsw exerted no more profound effects on the induction of γ-secretase proteins and its activity than did transfection with hPEN-2 alone. Thus, PEN-2 overexpression may facilitate assembly into the more active γ-secretase complex, and may also induce an increase in activity, thus affecting Aβ-42 production.     

Serum decreases the size of Metafectene-and Genejammer-DNA complexes but does not affect significantly their transfection activity in SCCVII murine squamous cell carcinoma cells

Cationic liposome-DNA (lipoplexes) or polymer-DNA (polyplexes) complexes have been used to deliver therapeutic genes, both in vitro and in vivo. However, gene transfer by these non-viral vectors is usually inhibited by biological milieu. A relatively high efficiency of transfection could be achieved in human oral cancer cells transfected with the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in the presence of 60% fetal bovine serum (FBS) (Konopka et al., Cell. Mol. Biol. Lett. 10 (2005) 455–470). Here, we examined the efficacy of these vectors to deliver β-galactosidase (β-gal), luciferase and Herpes Simplex Virus thymidine kinase (HSV-tk) genes to SCCVII murine squamous cell carcinoma cells, which are used to generate an orthotopic murine model of oral cancer. We also evaluated the hydrodynamic size and zeta potential of the vectors and the effect of FBS and mouse serum (up to 60%) on the size of Metafectene and GeneJammer complexes with the pCMV.Luc plasmid. Our results indicate that Metafectene and GeneJammer are highly effective in transfecting SCCVII cells. Approximately 60–70% of SCCVII cells transfected with pCMV.lacZ were positive for β-gal staining. The expression of β-galactosidase was essentially not affected by serum. Mouse serum (20–60%) reduced both Metafectene-and GeneJammer-mediated luciferase expression by ∼30–45%, while FBS did not affect transfection efficiency. The delivery of the HSV-tk gene by Metafectene or GeneJammer in the presence of 0% or 60% FBS, followed by GCV treatment for 6 days, resulted in over 90% cytotoxicity. The mean diameters of the DNA complexes of Metafectene and GeneJammer decreased significantly as a function of the serum concentration. The reduction in the size of the lipoplexes and polyplexes by serum was essentially not inhibitory to transfection of SCCVII cells. This is in contrast to previous hypotheses that serum-induced decrease in the size of lipoplexes is the primary cause of serum inhibition of transfection.     

Fabrication and Optimization of Linear PEI-Modified Crystal Nanocellulose as an Efficient Non-Viral Vector for In-Vitro Gene Delivery

Background:One of the major challenges in gene therapy is producing gene carriers that possess high transfection efficiency and low cytotoxicity (1). To achieve this purpose, crystal nanocellulose (CNC) -based nanoparticles grafted with polyethylenimine (PEI) have been developed as an alternative to traditional viral vectors to eliminate potential toxicity and immunogenicity.Methods:In this study, CNC-PEI10kDa (CNCP) nanoparticles were synthetized and their transfection efficiency was evaluated and compared with linear cationic PEI10kDa (PEI) polymer in HEK293T (HEK) cells. Synthetized nanoparticles were characterized with AFM, FTIR, DLS, and gel retardation assays. In-vitro gene delivery efficiency by nano-complexes and their effects on cell viability were determined with fluorescent microscopy and flow cytometry.Results:Prepared CNC was oxidized with sodium periodate and its surface cationized with linear PEI. The new CNCP nano-complex showed different transfection efficiencies at different nanoparticle/plasmid ratios, which were greater than those of PEI polymer. CNPC and Lipofectamine were similar in their transfection efficiencies and effect on cell viability after transfection.Conclusion:CNCP nanoparticles are appropriate candidates for gene delivery. This result highlights CNC as an attractive biomaterial and demonstrates how its different cationized forms may be applied in designing gene delivery systems.Key Words: Crystal Nanocellulose, Gene transfection, Nanoparticle, Nano-complex     

Species-specific expression of sialosyl-Lex on polymorphonuclear leukocytes (PMN), in relation to selectin-dependent PMN responses

Sialosyl-Le^x (SLe^x) and its positional isomer sialosyl-Le^a are the epitopes recognized by the lectin domain of E- and P-selectins. Expression of SLe^x in polymorphonuclear leukocytes (PMN) plays an important role in recruitment of these cells at sites of inflammation through activation of selectins. We studied expression of SLe^x in PMN of seven mammalian species in comparison with that in humans. Only PMN of humans (no other species) expressed SLe^x or other lacto-series epitopes such as Le^x or Le^y. The observed absence of these epitopes in rat PMN seems inconsistent with recent reports that the lung inflammation process in a rat model is inhibited by perfusion of SLe^x oligosaccharide (Mulligan MS,et al. (1993a)Nature 364:149; (1993b)J Exp Med 178:623). Rat selectins may be able to recognize SLe^x, even though this epitope is absent in rat PMN.Abbreviations FITC fluorescein isothiocyanate - mAb monoclonal antibody - PMN polymorphonuclear leukocytes - SLe^a sialosyl-Le^a antigen - SLe^x sialosyl-Le^x antigen     

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo

Background

Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature.

Methods and results

We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino‐sugar‐based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin‐cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo.

Conclusions

These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

基于脂蛋白P80的滑液支原体抗体ELISA检测方法的建立及应用

【目的】研究滑液支原体(Mycoplasma synoviae, MS)脂蛋白P80的免疫反应性及其在MS血清抗体ELISA检测中的应用。【方法】对MSP80的氨基酸序列进行生物信息学分析、原核表达和纯化,并用免疫印迹法分析其与6种不同MS分离株阳性血清的免疫反应性以及与其他禽病原血清的交叉反应性;运用纯化的MS P80表达蛋白作为包被抗原建立了MS血清抗体的间接ELISA检测方法,对其敏感性和重复性进行检测;比较检测了与美国爱德士检测试剂盒对50份临床血清样品的阳性符合率。【结果】生物信息学分析预测MS P80蛋白为脂蛋白且含有信号肽,其在MS种内同源性高达98%-100%,与其他种属P80蛋白同源性在25%-34%之间,成功表达和纯化了MS P80重组蛋白(rMS P80);Western blotting分析表明纯化的rMS P80具有良好的免疫反应性和特异性;运用rMS P80建立的MS血清ELISA抗体检测方法可对不同株MS阳性血清进行抗体效价检测,而对其他禽病原阳性血清均无交叉反应性;该检测方法的批内变异系数小于5%,批间变异系数小于10%,重复性良好;与美国IDEXX检测试剂盒比较,本文建立的ELISA抗体检测方法敏感性更高,阳性符合率为75%,阴性符合率为89.47%,总样本符合率为86%。【结论】MS P80具有较好的免疫反应性、种内保守性和种间特异,并且可用作MS抗体检测的靶标抗原。     

光唇鱼源无乳链球菌的分离鉴定及遗传特征

【目的】为查明浙江养殖光唇鱼大量死亡的病原,了解病原的遗传特征。【方法】本工作对患病光唇鱼进行病原分离,结合形态特征、生理生化特性和16S rRNA基因序列同源性,对分离菌株进行鉴定;采用人工回感试验确定其病原性,并对分离株的血清型、多位点序列分型(multilocus sequence typing,MLST)、毒力基因型和表面蛋白抗原基因型等遗传特征进行分析;此外,还测试了菌株的药敏特性。【结果】从患病光唇鱼体中分离得到优势菌株ACRO-0708,为革兰氏阳性球菌,不溶血,分子与生化鉴定为无乳链球菌(Streptococcus agalactiae);人工感染试验证实其对光唇鱼有较强的致病性,LD50为6.47×10~3CFU/g,属于血清型Ⅰb和MLST型ST261,毒力基因型为sip~+bibA~+cfb~+hylB~+iagA~+fbsA~+fbsB~+bac~–bca~–cylE~–scpB~–lmb~–,不携带所检测的6种表面蛋白基因。药敏试验结果显示,对青霉素、氨苄西林等8种药物较敏感,对氯霉素、复方新诺明等7种药物耐药。【结论】引起浙江养殖光唇鱼死亡的病原菌为无乳链球菌,其分子特征与水产动物主要流行的无乳链球菌株具有显著差异,生产中可选用氨苄西林、氟苯尼考等药物进行防治。     

Isolation of the CD7 gene from the DNA of transfected L cells

Using DNA from L cells which expressed high levels of the CD7 (Leu-9 or HuLy-m2) antigen obtained after two cycles of transfection, a genomic library was constructed in the lambda phage Charon 4A. Recombinant clones containing the gene coding for this antigen were identified by first screening the library with both the HSVtk gene and a probe detecting the human repetitive (Alu) sequences. DNA from 10 tk⁺ and 12 Alu⁺ recombinant clones was used to transfect L cells which were analyzed for the cell-surface expression of CD7 either early (48–72 h posttransfection) or later when hypoxanthine aminopterin thymidine-resistant colonies were obtained. Transfection with either Alu⁺ or tk⁺ recombinant phages led to transient early expression of CD7, and stable CD7⁺ transfectants were also established. Thus the CD7 gene has been isolated in a number of clones in association with either the Alu repetitive sequence or with the HSV-tk gene; the insert size in one of the genomic clones was 13.5 kb.