非病毒转基因载体的研究进展

高效的转基因载体是基因释放系统的核心。非病毒转基因载体具有低免疫原性和易制备等特性。本就近年来非病毒转基因载体的发展作一综述。     

非病毒转基因载体的研究进展

高效的转基因载体是基因释放系统的核心。非病毒转基因载体具有低免疫原性和易制备等特性。本文就近年来非病毒转基因载体的发展作一综述     

非病毒载体聚乙烯亚胺转基因因素的优化

聚乙烯亚胺属于阳离子多聚物,可浓缩DNA作为转基因非病毒载体。但其转染影响变量较多,如果不能充分控制将不能得到重复的、良好的结果。这里测定了聚乙烯亚胺转基因效率的影响因素,为合成更复杂的人工转基因载体创造条件。我们通过聚乙烯亚胺转染编码β-半乳糖苷酶的pSVβ质粒到CO S-7和N IH 3T 3细胞中,测定质粒因素、血清、细胞密度、操作方式以及转染复合物的保存因素对聚乙烯亚胺转基因效率的影响。结果表明:质粒中生物活性抑制剂明显降低转染效率,通过截流分子量3000或10000的超滤可以除去生活性抑制剂;断裂的质粒降低转染效率;培养液中的血清、白蛋白降低转染效率;细胞密度影响转染效率;PE I/DNA复合物与细胞作用8h后吸去,转染效果最优。冻存显著降低PE I/DNA转染复合物转染效率。聚乙烯亚胺可以作为合成新型非病毒载体的骨架,但必须控制有关因素才能发挥最佳的和可重复性的转染结果。     

siRNA非病毒纳米载体研究进展

当前,小分子干扰RNA的递送方式主要包括病毒载体载送、化学修饰载送、显微注射载送、非病毒纳米载体载送。非病毒纳米载体载送与其它载送方式相比具有简便、安全、易被患者接受等优点,但由于siRNA易水解以及被细胞摄取能力较差等原因,细胞呈递效率普遍较低。因此,近年来旨在优化siRNA、增加其应用范围而对其进行修饰的研究越来越多。本文就近期siRNA非病毒纳米载体研究进行综述,为其临床应用提供理论依据。     

聚乙二醇在非病毒转基因载体中的应用

非病毒转基因载体在基因治疗领域的应用日趋广泛,但目前开发的非病毒转基因载体直接体内应用由于存在诸多问题而受到限制。受药物和蛋白质聚乙二醇化后能增加目的药物和蛋白质稳定性,延长体内半衰期,降低毒副作用等效应的启发,近年有许多研究将聚乙二醇化应用于非病毒转基因载体,取得了一定的效果。     

基因治疗中的非病毒载体

添加、修复或替换基因从而达到直接排除病因是基因治疗的目的，也是用于治疗可遗传和获得性疾病的治疗方法。它包括目的基因、载体和靶细胞三个方面，其中载体在整个转染过程中起着关键的作用。非病毒载体是该研究领域的热点之一，虽然转染效率不如病毒载体，但其无毒、无免疫反应、性质可调且制备方便。本文主要就非病毒载体的转染机理及优化策略作一综述。     

非病毒型纳米载体在基因治疗中的研究现状及展望

近10年来，新型非病毒载体在基因治疗中日益受到欢迎。其主要代表为纳米载体，具有无毒性及免疫原性的优势，已作为高效阳离子载体用于基因转移。体外基因转移实验表明，纳米载体的基因转移率高于普通脂质体及其它阳离子多聚体，如多聚氮丙及聚赖氨酸。本对纳米载体的结构特点，性能，基因转移机制进行综述，并将其在体内外基因转移效率与其它非病毒载体作以比较。     

基因治疗新型病毒载体研究进展

基因转移载体是基因治疗能否进入临床应用的关键之一,本文就不断出现的新型病毒载体的研究方面的最新进展包括载体的构建、改造、表达调控和应用作了综述和评价。     

基因治疗新型病毒载体研究进展

基因转移载体是基因治疗能否进入临床应用的关键之一,本文就不断出现的新型病毒载体研究方面的最新进展包括载体的构建、改造、表达调控和应用作了综述和评价。     

非病毒型纳米载体在基因治疗中的研究现状及展望

近 10年来 ,新型非病毒载体在基因治疗中日益受到欢迎。其主要代表为纳米载体 ,具有无毒性及免疫原性的优势 ,已作为高效阳离子载体用于基因转移。体外基因转移实验表明 ,纳米载体的基因转移率高于普通脂质体及其它阳离子多聚体 ,如多聚氮丙啶及聚赖氨酸。本文对纳米载体的结构特点、性能、基因转移机制进行综述 ,并将其在体内外基因转移效率与其它非病毒载体作以比较     

Gene delivery using dendrimer-entrapped gold nanoparticles as nonviral vectors

Development of highly efficient nonviral gene delivery vectors still remains a great challenge. In this study, we report a new gene delivery vector based on dendrimer-entrapped gold nanoparticles (Au DENPs) with significantly higher gene transfection efficiency than that of dendrimers without AuNPs entrapped. Amine-terminated generation 5 poly(amidoamine) (PAMAM) dendrimers (G5.NH₂) were utilized as templates to synthesize AuNPs with different Au atom/dendrimer molar ratios (25:1, 50:1, 75:1, and 100:1, respectively). The formed Au DENPs were used to complex two different pDNAs encoding luciferase (Luc) and enhanced green fluorescent protein (EGFP), respectively for gene transfection studies. The Au DENPs/pDNA polyplexes with different N/P ratios and compositions of Au DENPs were characterized by gel retardation assay, light scattering, zeta potential measurements, and atomic force microscopic imaging. We show that the Au DENPs can effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines as demonstrated by both Luc assay and fluorescence microscopic imaging of the EGFP expression. The transfection efficiency of Au DENPs with Au atom/dendrimer molar ratio of 25:1 was at least 100 times higher than that of G5.NH₂ dendrimers without AuNPs entrapped at the N/P ratio of 2.5:1. The higher gene transfection efficiency of Au DENPs is primarily due to the fact that the entrapment of AuNPs helps preserve the 3-dimensional spherical morphology of dendrimers, allowing for more efficient interaction between dendrimers and DNA. With the less cytotoxicity than that of G5.NH₂ dendrimers demonstrated by thiazoyl blue tetrazolium bromide assay and higher gene transfection efficiency, it is expected that Au DENPs may be used as a new gene delivery vector for highly efficient transfection of different genes for various biomedical applications.     

Enzyme-synthesized poly(amine-co-esters) as nonviral vectors for gene delivery

A family of biodegradable poly(amine-co-esters) was synthesized in one step via enzymatic copolymerization of diesters with amino-substituted diols. Diesters of length C(4) -C(12) (i.e., from succinate to dodecanedioate) were successfully copolymerized with diethanolamines with either an alkyl (methyl, ethyl, n-butyl, t-butyl) or an aryl (phenyl) substituent on the nitrogen. Upon protonation at slightly acidic conditions, these poly(amine-co-esters) readily turned to cationic polyelectrolytes, which were capable of condensing with polyanionic DNA to form nanometer-sized polyplexes. In vitro screening with pLucDNA revealed that two of the copolymers, poly(N-methyldiethyleneamine sebacate) (PMSC) and poly(N-ethyldiethyleneamine sebacate) (PESC), possessed comparable or higher transfection efficiencies compared with Lipofectamine 2000. PMSC/pLucDNA and PESC/pLucDNA nanoparticles had desirable particle sizes (40-70 nm) for cellular uptake and were capable of functioning as proton sponges to facilitate endosomal escape after cellular uptake. These polyplex nanoparticles exhibited extremely low cytotoxicity. Furthermore, in vivo gene transfection experiments revealed that PMSC is a substantially more effective gene carrier than PEI in delivering pLucDNA to cells in tumors in mice. All these properties suggest that poly(amine-co-esters) are promising nonviral vectors for safe and efficient DNA delivery in gene therapy.     

Development of retroviral vectors as safe, targeted gene delivery systems

The transfer of genes of potential therapeutic benefit is presently being attempted in the clinic to treat a number of genetic and virally induced diseases. Many of these protocols use retroviral vectors derived from murine leukemia retroviruses as gene delivery systems. Although these viral delivery systems are well suited for this purpose, a number of their characteristics, some of which are discussed here, are still troublesome. Future retroviral vectors will incorporate nonretroviral features and will be tailored to desired needs for specific uses. These vectors will be safer, more efficient, and targeted in their delivery. Further, expression of the therapeutic genes carried will be limited to the specific target cell type. Some of the recent advances that have been made towards this goal are reviewed here.     

非病毒基因载体材料的研究进展

非病毒材料可成为基因治疗中的基因载体,使目的基因持续有效地表达。非病毒基因载体主要有脂质体、人工合成聚合物载体、天然聚合物载体、局部基因释放载体等。其中壳聚糖及其衍生物是一种优良的基因释放载体,局部基因释放载体技术将基因治疗与组织工程结合起来,在组织修复与重建方面将发挥重要作用。     

Cationic star polymers consisting of alpha-cyclodextrin core and oligoethylenimine arms as nonviral gene delivery vectors

A series of novel cationic star polymers were synthesized by conjugating multiple oligoethylenimine (OEI) arms onto an alpha-cyclodextrin (alpha-CD) core as nonviral gene delivery vectors. The molecular structures of the alpha-CD-OEI star polymers, which contained linear or branched OEI arms with different chain lengths ranging from 1 to 14 ethylenimine units, were characterized by using size exclusion chromatography, 13C and 1H NMR, and elemental analysis. The alpha-CD-OEI star polymers were studied in terms of their DNA binding capability, formation of nanoparticles with plasmid DNA (pDNA), cytotoxicity, and gene transfection in cultured cells. All the alpha-CD-OEI star polymers could inhibit the migration of pDNA on agarose gel through formation of complexes with pDNA, and the complexes formed nanoparticles with sizes ranging from 100 to 200 nm at N/P ratios of 8 or higher. The star polymers displayed much lower in vitro cytotoxicity than that of branched polyethylenimine (PEI) of molecular weight 25K. The alpha-CD-OEI star polymers showed excellent gene transfection efficiency in HEK293 and Cos7 cells. Generally, the transfection efficiency increased with an increase in the OEI arm length. The star polymers with longer and branched OEI arms showed higher transfection efficiency. The best one of the star polymers for gene delivery showed excellent in vitro transfection efficiency that was comparable to or even higher than that of branched PEI (25K). The novel alpha-CD-OEI star polymers with OEI arms of different chain lengths and chain architectures can be promising new nonviral gene delivery vectors with low cytotoxicity and high gene transfection efficiency for future gene therapy applications.     

Poly(ethylene imine)-g-chitosan using EX-810 as a spacer for nonviral gene delivery vectors

Polyelectrolyte complexes have been widely studied as gene carriers in recent years. In this study, poly (ethylene imine) was grafted onto chitosan (PEI-g-CHI) as a nonviral gene carrier in order to improve the water solubility as well as the inherent transfection efficiency of chitosan. We present a novel method to conjugate the amine or hydroxyl groups of chitosan (CHI) and the amine groups of PEI through opening the epoxide rings of ethylene glycol diglycidyl ether (EX-810), which also brings the merits as mentioned in PEGylation chemistry. The degree of substitution of PEI onto CHI was characterized by NMR. The preliminarily cellular mechanisms, from the cellular entry to the endosomal release, were investigated by the correlations among the physicochemical properties of the DNA-polymer complexes, the buffering capacity of the modified polymer, the cytotoxicity, and the efficiency of the transgene expression. The cytotoxicity assayed by MTT shows that cell viability of PEI-g-CHI is higher than CHI especially noticeable at high concentrations using human kidney 293T cells. The efficiency of transgene expression and the amount of intracellular plasmid were monitored using green fluorescent protein (GFP) and visualized by fluorescence microscopy. The transfection efficiency of PEI-g-CHI/DNA polyplex is significantly better than CHI/DNA polyplex when using the weight ratios higher than 2.5.     

超声基因药物传递新方法的研究与评估

目的:开发超声裸基因药物传递与治疗新方法,研究适合超声基因传递的安全计量与参数,进行安全性评价,建立超声裸基因传递系统。方法:建立一套经水听器与声功率计标定的低频超声基因传递实验系统(35·1K),用不同的计量对兔、鸡、S180肿瘤细胞悬液进行暴露,提取最佳基因传递参数。用绿色荧光蛋白作为传递基因进行传递实验。用荧光法、分光光度法、扫描电镜法、激光共聚焦扫描法和流式细胞术评估实验结果。结果:最佳传递参数下低频超声有效地将GFP基因传入了S180细胞,未见明显的细胞损伤与细胞毒性,转染率为35·83%(±2·53,n=6),并经活体证明细胞功能正常。90%细胞存活点的能量积累E是最佳传递参数(E=3·56±0·06),80%细胞存活点的E值是损伤阈值(E=59·67±3·54)。GFP的表达明显比病毒载体法强(P<0·001)。结论:超声可安全地用于基因传递和治疗,其基因表达和摄入量取决于90%细胞存活点的能量积累E。它可用作控制因子与其它参数结合控制传递效应。从结果证明平稳空化产生基因传递效应,瞬间空化造成细胞损伤。     

Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting

The application of non-viral gene delivery vectors is often accompanied with the poor correlation between transfection efficiency and the safety profiles of vectors. Vectors with high transfection efficiencies often suffer from high toxicities, making it unlikely to improve their efficiencies by increasing the DNA dosage. In the current study, we developed a ternary complex system which consisted of a highly membrane-active cationic helical polypeptide (PVBLG-8), a low-toxic, membrane-inactive cationic helical polypeptide (PVBLG-7) capable of mediating mannose receptor targeting, and DNA. The PVBLG-7 moiety notably enhanced the cellular uptake and transfection efficiency of PVBLG-8 in a variety of mannose receptor-expressing cell types (HeLa, COS-7, and Raw 264.7), while it did not compromise the membrane permeability of PVBLG-8 or bring additional cytotoxicities. Because of the simplicity and adjustability of the self-assembly approach, optimal formulations of the ternary complexes with a proper balance between membrane activity and targeting capability were easily identified in each specific cell type. The optimal ternary complexes displayed desired cell tolerability and markedly outperformed the PVBLG-8/DNA binary complexes as well as commercial reagent Lipofectamine™ 2000 in terms of transfection efficiency. This study therefore provides an effective and facile strategy to overcome the efficiency-toxicity poor correlation of non-viral vectors, which contributes insights into the design strategy of effective and safe non-viral gene delivery vectors.     

成年大鼠胰岛的体外基因转染

目的 探讨携带外源基因的慢病毒在体外有效感染胰岛及外源基因在胰岛中的表达，为通过移植前向胰岛细胞转入特定的免疫调节分子基因诱导胰岛移植物耐受奠定基础。方法 将目的基因CTLA4Ig导入慢病毒载体pWPTS，构建成pWPTS-CTLA4Ig载体。用磷酸钙沉淀法将pWPTS-EGFP、pWPTS-CTLA4Ig分别和其辅助载体pMD2．G、pCMVΔ8．92共转染293T细胞，收获病毒上清液，测定病毒滴度后感染新分离的胰岛。通过Western Blot测定胰岛培养上清液中CTLA4Ig蛋白的表达。结果 ①成功构建了携带CTLA4Ig基因的慢病毒载体pWPTS-CTLA4Ig；②包装产生的慢病毒Lenti-EGFP、Lenti-CTLA4Ig在体外可以感染胰岛，其中在Lenti-EGFP慢病毒感染的胰岛观察到了绿色荧光，及在Lenti-CTLA4Ig慢病毒感染的胰岛培养上清液中检测到了CTLA4Ig蛋白的表达。结论 慢病毒在体外可以有效感染大鼠胰岛，且携带的外源基因可以在胰岛细胞中稳定表达，其中Lenti-CTLA4Ig慢病毒感染的胰岛为进行胰岛移植并诱导体内特异的胰岛移植物耐受奠定了基础。