基因治疗用质粒DNA大规模制备及质量控制

基因治疗需要数量可观的药学规格的质粒ＤＮＡ产品。目前大规模制备质粒ＤＮＡ存在许多有待解决的问题，如产量小、碱裂解缺乏重复性、大规模色谱分离纯化困难等。质粒ＤＮＡ最终产品的质量涉及到制备的整个过程，应在研究阶段、生产阶段和最终产品等不同阶段加以控制。     

用于基因治疗的药物级质粒DNA的大规模生产：问 …

基因疗法是用于预防及治疗癌症、艾滋病和囊性纤维变性等疾病的有效方法。给予治疗基因的方法之一是直接注射露或脂质体包裹的质粒ＤＮＡ,但需要大量质粒ＤＮＡ。当前在基因治疗用药物质粒的ＤＮＡ的大规模生产工艺的设计及操作中还存在一些问题和障碍。     

菌体中大量抽提质粒DNA方法的改进

目的　建立简便、快捷的大量制备质粒的实验方法。方法　以文献报道方法为依据 ,将传统的质粒小量抽提的实验方法与大量抽提的实验方法有机结合 ,以琼脂糖凝胶电泳的方法证明了使用改进后方法的可行性。结果　使用该法可以一次大量地制备质粒DNA ( 690± 2 19) μg ,提取效力为 ( 1 4± 0 4) μg/ml菌液 ,并可直接用于酶切。结论　改进后的实验方法更加简便、快捷、高效 ,为分子生物研究中质粒的大量制备提供了更加实用的实验方法     

基因治疗和DNA疫苗质粒DNA的下游加工技术

随着基因治疗和DNA疫苗的快速发展，研究人员对大量制备超螺旋质粒DNA方法的研究兴趣也随之增加。文中综述了大规模质粒DNA的纯化工艺，讨论了生产符合规格的终产品的原理和方法。     

从药学观点出发的质粒DNA分析

基因治疗和基于多核苷酸疫苗出现,使质粒ＤＮＡ可以作为一种药物来使用,当前,确定这种药物的特性和效力还必须采用生物学的方法,但在将来,基于微晶片的基因突变检测手段将与一系旬的色谱,电泳,流体力学以及光谱方法组合,从而得到精确的测定,本文综述了确定药物质粒ＤＮＡ性质的多种方法,并讨论了质粒的化学稳定性问题。     

无抗生素质粒DNA扩增系统的探讨

质粒DNA疫苗在很多方面克服了传统疫苗和病毒载体疫苗的缺陷,本文对无抗生素质粒DNA扩增系统发展现状进行作一简要概述.     

建立一种全质粒DNA序列测定方法

质粒DNA的序列测定是现代分子生物学的重要技术,我们改进了以往质粒DNA序列测定的操作,建立一种新的全质粒DNA序列测定方法,7个质粒DNA测定结果表明:该方法结果稳定可靠,快速简便,值得推广应用。     

使用碱裂解法快速提取药材DNA方法的研究

目的:建立药材DNA快速提取方法。方法:使用碱裂解缓冲液提取药材DNA,考察提取液配方组成和中和剂种类对药材DNA纯度、浓度的影响。利用优化提取液提取了144个中药材DNA,动物药使用COⅠ、植物药使用psbA-trnH通用引物进行PCR扩增。结果:0.5 mol.L-1氢氧化钠、1%PVP和1%Triton×100具有最佳的DNA提取效果。对144个中药材进行PCR扩增,利用琼脂糖凝胶电泳可检测到123个中药材DNA的PCR产物。碱裂解法可用于快速提取蛇类药材DNA,并成功用于乌梢蛇、金钱白花蛇的分子鉴定。结论:优化的碱裂解法可用于提取中药材基因组DNA,其提取时间可控制在10 min左右,且避免了酚、三氯甲烷等对人体有毒试剂的使用,对药材快速分子鉴定体系的建立将发挥重要作用。     

简化的质粒癌基因DNA制备方法介绍

癌基因的活化或抗癌基因(或称肿瘤控制基因)的失活在致癌过程中的作用愈显重要。目前有30多种含质粒癌基因的大肠杆菌菌种已成为商品可购得。质粒癌基因DNA的制备是致癌机理研究中制备相应DNA探针的必要手段。本文介绍简化     

A Scalable,Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA

No HeadingPurpose. A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy.Methods. Liposomes encapsulating plasmid DNA were formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner. Combining DNA-buffer and lipid-ethanol flow streams in a T-shaped mixing chamber resulted in instantaneous dilution of ethanol below the concentration required to support lipid solubility. The resulting DNA-containing liposomes were further stabilized by a second stepwise dilution.Results. Using this method, monodisperse vesicles were prepared with particle sizes less than 200 nm and DNA encapsulation efficiencies greater than 80%. In mice possessing Neuro 2a tumors, SPLP demonstrated a 13 h circulation half-life in vivo, good tumor accumulation and gene expression profiles similar to SPLP previously prepared by detergent dialysis. Cryo transmission electron microscopy analysis showed that SPLP prepared by stepwise ethanol dilution were a mixed population of unilamellar, bilamellar, and oligolamellar vesicles. Vesicles of similar lipid composition, prepared without DNA, were also <200 nm but were predominantly bilamellar with unusual elongate d morphologies, suggesting that the plasmid particle affects the morphology of the encapsulating liposome. A similar approach was used to prepare neutral egg phosphatidylcholine:cholesterol (EPC:Chol) liposomes possessing a pH gradient, which was confirmed by the uptake of the lipophilic cation safranin O.Conclusions. This new method will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies. Additionally, this method now allows for the acceleration of SPLP formulation development, enabling the rapid development and evaluation of novel carrier systems.     

Plasmid DNA vaccines

Genes that code for the production of protein antigens have been cloned and recombined with plasmids. Gene-plasmid constructs have been amplified in a bacterial host, purified and administered to a mammalian host. The gene is expressed in the host and the antigen that is produced induces an immune response. These so-called DNA vaccines have been prepared for a number of infectious agents, some tumors and some allergens, and were shown to be efficacious in animal studies. Clinical trials for some of these vaccines are underway. Advantages of using a DNA vaccine include the abilities to favor a T helper-1 or a T helper-2 lymphocyte response and to induce a cytotoxic T-lymphocyte response. Moreover, some reports have indicated that they produce long-lasting immunity. DNA vaccines might be used in situations where no effective vaccine is available for a disease. However, their use might not be risk-free. Further research in this field is needed to determine their efficacy and to identify the risks involved in using them.     

Polyethylene Glycol-Conjugated Copolymers for Plasmid DNA Delivery

No Heading Polymeric gene delivery systems have been developed as an alternative for viral gene delivery systems to overcome the problems in the use of viral gene carriers. Polymeric carriers have many advantages as gene carriers such as low cytotoxicity, low immunogenicity, moderate transfection efficiency, no size-limit, low cost, and reproducibility. In the efforts to develop safe and efficient polymeric gene carriers, polyethylene glycol (PEG) has widely been used because of its excellent characteristics. PEG-conjugated copolymers have advantages for gene delivery: 1) The PEG-conjugated copolymers show low cytotoxicity to cells in vitro and in vivo, 2) PEG increases water-solubility of the polymer/DNA complex, 3) PEG reduces the interaction of the polymer/DNA complex with serum proteins and increases circulation time of the complex, 4) PEG can be used as a spacer between a targeting ligand and a cationic polymer. A targeting ligand at the end of a PEG chain is not disturbed by the interaction of a cationic polymer with plasmid DNA, and the PEG spacer increases the accessibility of the ligand to its receptor. In this review, PEG copolymers as gene carriers are introduced, and their characteristics are discussed.     

Identifying Stabilizers of Plasmid DNA for Pharmaceutical Use

To better address the need for developing stable formulations of plasmid DNA-based biopharmaceuticals, 37 compounds from a generally regarded as safe library were examined for their potential use as stabilizers. A plasmid DNA-based therapeutic vaccine, BHT-DNA?, was used as a model system. Initial studies were performed to compare the biophysical properties of BHT-DNA? plasmid from bulk drug substance and finished drug product. An agarose gel electrophoresis-based assay was then employed in excipient compatibility studies for the drug product by monitoring supercoiled plasmid DNA content in various formulations. After incubation at 40°C for 30 days, eight out of the 37 excipients tested were able to better retain the supercoil content compared to the control. Sodium citrate appeared to be the most effective stabilizer and its protective capability plateaued at an ionic strength of about 0.4. Several other excipients including malic acid, ethanol, and Pluronic F-68 were also identified as promising stabilizers for BHT-DNA? plasmid DNA. Additionally, compounds, including ferrous chloride, ascorbic acid, human serum albumin, and PEG 1000, which significantly destabilized the supercoiled plasmid DNA were identified. These data may also be applicable to other plasmid DNA-based pharmaceuticals for storage stability improvement, due to chemical and structural similarities of these macromolecules.     

Oral Plasmid DNA Delivery Systems for Genetic Immunisation

The use and optimisation of plasmid DNA delivery systems for the purposes of eliciting transgene specific immune responses to orally administered DNA encoded antigen represents a significant challenge. Here, we have outlined a multicomponent polymer modified liposomal delivery system that offers potential for oral administration of plasmid DNA. It is shown that the polymer/liposome formulated DNA is able to elicit markedly enhanced transgene specific cytokine production following in vitro restimulation of splenocytes with recombinant antigen. This is discussed with reference to recent publications and the potential of plasmid DNA delivery systems for the purposes of genetic immunisation, as reported in selected literature, is assessed.     

重组水蛭素Ⅲ的中试工艺研究

从大肠杆菌中大规模纯化重组水蛭素Ⅲ,进行重组水蛭素Ⅲ中试发酵工艺和纯化中试工艺研究,连续大量纯化3批,并对纯化终产品进行鉴定。通过菌种活化,一级、二级种子液制备,30L发酵罐补料-批式发酵水蛭素产量达到6000ATU/ml,发酵液经还原性SDS-PAGE分析表明在14000u处有单一条带,为重组水蛭素Ⅲ二聚体。经大孔树脂层析、DEAE-纤维素层析、制备型反相高效液相层析等3步纯化后重组水蛭素Ⅲ的比活达8270ATU/mg。经分析型反相高效液相色谱分析,纯度达95%以上,总收率达到39%以上。通过实验建立了稳定的发酵和纯化工艺流程。     

亚单位流感疫苗中裂解剂检测方法的改进

目的 改进检测亚单位流感疫苗中裂解剂(壬苯醇醚-9)含量的HPLC方法.方法 以C4柱(Thermo BioBasic-4)代替药典使用的C18柱(Thermo BDS Hypersil C18),改变相应流动相比例.结果 C4柱保留时间仅3.27～3.34min,杂峰少,易积分,在5～2500tg·mL-1范围内线性关系良好,回收率约为100％,日内精密度和日间精密度均小于3％.结论 C4柱与C18柱相比更快速准确,更适用于实验室对裂解剂含量的常规检测.     

Pharmacokinetics of Plasmid DNA in the Rat

Purpose. The pharmacokinetics of plasmid DNA after IV bolus administration in the rat by following supercoiled (SC), open circular (OC), and linear (L) pDNA forms of the plasmid.Methods. SC, OC, and L pDNA were injected at 2500, 500, 333, and 250 g doses. The concentrations in the bloodstream of OC and L pDNA were monitored.Results. SC pDNA was detectable in the bloodstream only after a 2500 g dose, and had a clearance of 390(±50) ml/min and V_d of 81(±8) ml. The pharmacokinetics of OC pDNA exhibited non-linear characteristics with clearance ranging from 8.3(±0.8) to 1.3(±0.2) ml/min and a V_d of 39(±19) ml. L pDNA was cleared at 7.6(±2.3) ml/min and had a V_d of 37(±17) ml. AUC analysis revealed that 60(±10) % of the SC was converted to the OC form, and nearly complete conversion of the OC pDNA to L pDNA. Clearance of SC pDNA was decreased after liposome complexation to 87(±30) ml/min. However the clearance of OC and L pDNA was increased relative to naked pDNA at an equivalent dose to 37(±9) ml/min and 95(±37) ml/min respectively.Conclusions. SC pDNA is rapidly metabolized and cleared from the circulation. OC pDNA displays non-linear pharmacokinetics. Linear pDNA exhibits first order kinetics. Liposome complexation protects the SC topoform, but the complexes are more rapidly cleared than the naked pDNA.     

壳聚糖制备工艺改进

报道从虾蟹甲壳制备壳聚糖的改进工艺,比传统的生产工艺缩短了制备周期,节约了能源和原材料。     

The Effect of Lyophilization on Plasmid DNA Activity

The effect of lyophilization of plasmid DNA's ability to express an encoded protein was studied. Plasmid DNA, pRL-CMV expressing Renilla luciferase, was purified and stored in Tris-ethylenediaminetetraacetic acid (EDTA) buffer. Aliquots of the plasmid were lyophilized using analytical equipment, both alone and in the presence of carbohydrate. Samples were rehydrated and subject to functional and structural analyses. Analytical techniques included transfection efficiency in COS-1 cells, agarose gel electrophoresis, dimethylethylenediamine (DMED) assay for abasic sites, circular dichroism measurement, and UV spectroscopy. The lyophilization of pRL-CMV plasmid DNA resulted in a statistically significant loss of transfection efficiency (p < 0.05). Mono- and disaccharides could completely restore transfection efficiency. Agarose gel electrophoresis and the DMED assay demonstrated no change in gross plasmid structure or increase in abasic sites during lyophilization, respectively. Changes in DNA form, as measured by a change in ellipsisity, were observed on lyophilization. However, these changes were transient and were not shown to be responsible for loss of transfection efficiency. A hyperchromic effect was observed at 260 nm after lyophilization and could be reversed by the presence of carbohydrates. Lyophilization causes a decrease in plasmid DNA activity as measured by an in vitro transfection assay. Carbohydrates can ameliorate this decreased activity, which may be due to structural changes seen during the lyophilization process.