Enhancement of the efficiency of PEI/liposome transfection by magnetofectins formed via electrostatic self-assembly

One of the major challenges for successful gene therapy is improving the transfection efficiency of non-viral vectors. Magnetic nanoparticles (MNPs) have been developed as enhancers of non-viral vehicles. We prepared MNPs and modified them with polyethyleneimine (PEI), citric acid (CA) or carboxylmethyl-dextran (CMD). Both positively charged MNPs (MNPs@PEI) and negatively charged MNPs (MNPs@CA, MNPs@CMD) could spontaneously form transfection complexes (magnetofectins) with plasmid DNA and PEI/liposome via electrostatic self-assembly. Our results showed as-prepared magnetofectins apparently enhanced PEI/liposome transfection efficiency and/or gene expression level into COS-7 cells with reduced transfection time from 4 h to 15 min under a magnetic field in vitro. Meanwhile, the effect of magnetofection was cell line-dependant. These results suggest that charged MNPs could improve transfection efficiency for non-viral vectors by simply mixing with them and by exerting a magnetic force. Thus such MNPs provide a convenient platform for further applications of gene delivery.     

Recent progress in polymer-based gene delivery vectors

The gene delivery system is one of the three components of a gene medicine, which is the bottle neck of current gene therapy. Nonviral vectors offer advantages over the viral system of safety, ease of manufacturing, etc. As important nonviral vectors, polymer gene delivery systems have gained increasing attention and have begun to show increasing promising. In this review, the fundamental and recent progress of polymer-based gene delivery vectors is reviewed.     

心血管疾病的基因治疗

基因治疗在先天遗传性以及后天获得性心血管疾病治疗中均具有广阔的发展前景. 对心血管疾病致病机理的深入认识和疾病基因组学研究的发展, 进一步促进了临床前基因治疗的研究进展. 但基因治疗过程中存在的机体细胞免疫反应、外源基因表达水平不足、在体基因转导效率低下等因素都成为基因治疗临床应用转化的瓶颈. 近年来, 基因导入载体和基因组编辑技术的发展为上述问题的改善和解决提供了新的思路. 目前成族规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)/Cas9 基因组编辑技术已经成功应用于动物模型的在体基因编辑, 达到了显著改善血脂指标的疗效. 进一步研究体内组织特异和高效的基因导入方式, 提高基因编辑的靶向效率和特异性, 并建立全面有效的安全评估实验体系, 将推动基因治疗向临床应用的转化. 针对心血管疾病基因治疗中基因导入载体的研究以及CRISPR/Cas9 基因组编辑技术的应用展开讨论.     

肿瘤基因治疗载体的研究进展

对在肿瘤基因治疗中较常见的载体--病毒载体(痘苗病毒载体、腺病毒与腺病毒相关载体、单纯疱疹病毒和逆转录病毒)和非病毒载体(脂质体和稳定质粒--脂质颗粒)进行了介绍,同时对其存在的问题作了简要阐述,并对该研究前景进行了展望.     

用于高效磁转染的鱼精蛋白修饰的铁氧磁性纳米粒研究

 功能化铁氧磁性纳米粒在生物医学中应用广泛,可用于肿瘤磁感应热疗、磁共振成像（Magnetic Resonance Imaging,MRI）、药物输送及磁转染等方面。为了探讨鱼精蛋白功能化修饰的铁氧磁性纳米粒的制备及其作为基因载体在体外磁转染中的可行性,采用共沉淀法制备Fe3O4磁性纳米粒,经表面氨基化修饰后在其表面偶联鱼精蛋白。利用透射电镜、傅里叶红外光谱仪、zeta电位与粒度分析仪等,对磁性纳米粒进行形态、粒径及zeta电位分析等表征检测。共聚焦显微镜观察磁转染方法转染报告基因绿色荧光蛋白质粒pEGFP-N1进入HepG2细胞的表达,以真核转染试剂vigofect为对照。结果显示,实验中制备的磁性纳米粒粒径10nm左右,在交变磁场下具有良好的升温性能。鱼精蛋白功能化修饰磁性纳米粒后,其zeta电位进一步增大,更利于与DNA有效结合,在HepG2细胞系,其转染pEGFP-N1质粒的效率高于vigofect。研究表明,鱼精蛋白功能化修饰的铁氧磁性纳米粒可作为磁转染的有效载体,由于其同时具备在交变磁场下升温的性能,在基因治疗联合热疗的研究领域具有一定的应用价值。     

阳离子脂质体在基因治疗中的应用

介绍了阳离子脂质体的组成和结构特点以及作为基因载体在基因治疗方面的应用．阳离子脂质体转基因的机理研究表明：阳离子脂质体／基因复合物进入细胞的方式依赖于细胞的内吞作用或通过细胞的膜融合作用．     

使用病毒载体的罕见病基因疗法临床进展

 很大一部分的罕见病由遗传因素决定,难以用普通的小分子或大分子药物治愈,而基因治疗有望从根本上修正人体功能的缺失或异常,给罕见病患者带来改善生活质量的希望。目前许多基因疗法的临床试验正在开展,病毒载体是常用的基因递送方法,本文讨论了用于临床基因递送的多种病毒载体,包括腺相关病毒、逆转录病毒和慢病毒,重点列举了这些病毒在罕见病临床试验中的研究、应用和进展,评价了这些病毒的优缺点,并简述了基因疗法的研究方向及应用前景。     

Fabrication of PEI-Liposome Loaded Fish Gelatin Nanofibers Mats by Green Electrospinning

Polyethyleneimine （PEI） and cationic liposomes were widely used for gent delivery and the combination of PEI and liposome was reported to result in a higher efficiency of cell transfection in vitro. In recent years, better transfection was observed for the drug-loaded iiposome fixed on the tissue engineering scaffolds via embedding, surface adsorption or covalent grafting thus protected and bound by the scaffolds. In the present study, a novel PEI-liposome loaded fish gdatin composite nanofiber was successfully fabricated by a green electrosplnning process. The existence of PEI- liposome in the composite nanofibers was determined by Fourier transform infrared （ FTIR ） spectra, transmission electron microscopy （TEM）, and confoeal laser scanning microscopy （CLSM）. As shown by scanning electron microscopy （SEM）, the dectrospun composite nanofibers with uniform diameter were smooth and round, and the morphology of the fish gelatin fibers did not change significantly after the incorporation of PEI-liposomes. The transfection results in vitro suggest PEI-liposome loaded fish gelatin material may have a promising application in non-viral gene delivery systems.     

Pathway complexity in supramolecular polymerization

Self-assembly provides an attractive route to functional organic materials, with properties and hence performance depending sensitively on the organization of the molecular building blocks. Molecular organization is a direct consequence of the pathways involved in the supramolecular assembly process, which is more amenable to detailed study when using one-dimensional systems. In the case of protein fibrils, formation and growth have been attributed to complex aggregation pathways that go beyond traditional concepts of homogeneous and secondary nucleation events. The self-assembly of synthetic supramolecular polymers has also been studied and even modulated, but our quantitative understanding of the processes involved remains limited. Here we report time-resolved observations of the formation of supramolecular polymers from π-conjugated oligomers. Our kinetic experiments show the presence of a kinetically favoured metastable assembly that forms quickly but then transforms into the thermodynamically favoured form. Quantitative insight into the kinetic experiments was obtained from kinetic model calculations, which revealed two parallel and competing pathways leading to assemblies with opposite helicity. These insights prompt us to use a chiral tartaric acid as an auxiliary to change the thermodynamic preference of the assembly process. We find that we can force aggregation completely down the kinetically favoured pathway so that, on removal of the auxiliary, we obtain only metastable assemblies.     

Cationic polymeric nanoformulation: Recent advances in material design for CRISPR/Cas9 gene therapy

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat associated proteins 9) gene editing platform is a promising therapeutic tool for genetic disorders, due to its ability to manipulate the pathogenic gene in genomic level and to easily target specific gene by manipulating single-guide RNA. However, its successful delivery remains a challenge. Up to now, great efforts have been made to explore an effective strategy for CRISPR/Cas9 delivery. But among those delivery methods, physical methods are mainly operated on cultured cells thus limited to laboratorial use; viral vectors are hindered by fetal immunogenic and carcinogenic effects thus dubious in clinical application. Therefore, cationic polymeric vectors, with the ability to interact with CRISPR/Cas9 system to form a nanoformulation as a non-viral approach, are attracting increasing attentions, due to advantages such as well protection of cargos, less limitation in payload size, low immunogenicity or carcinogenicity, potential modifications for further functions, and ease in mass production. In this review, the recent discoveries on polymeric vectors utilized in delivery of CRISPR/Cas9 system will be summarized. With emphasis on advanced features of those polymeric vectors or their nanoformulations to meet the demands of different CRISPR/Cas9 delivery forms (plasmid, mRNA or protein), the detailed illustrations on their disease treatment applications, such as cancer, diabetes or antibiotic-resistant infections, will also be reviewed.     

复合环境离子强度对BDCP/DNA的粒径与转染效率的影响

为了探讨组装环境对基因载体/DN复合物的粒径和转染效果的影响,在三种不同离子浓度溶液体系(PBS, 5% 葡萄糖溶液, H₂O)中测量BDCP（biodegradable cationic polymer,一种生物可降解的阳离子聚合物）/DNA复合物粒径和结合力,进行了体外转染试验和毒性试验.结果显示,PBS最适合组装转染复合物,可取得更好的稳定性、最高的转染效率和较低细胞毒性、低溶血率;在5%葡萄糖溶液和水中组装的BDCP/质粒复合物结合力较弱,转染效率比较低.得出结论,BDCP/DNA粒径、结合力和基因转染效率受组装体系的离子强度影响.     

单纯疱疾病毒胸苷激酶基因的脑肿瘤基因治疗研究

单纯疱疾病毒胸苷激酶基因（HerpesSimplexVirus－ThymidineKinase,HSV－tk）,配合核苷酸类似物（NAS）GCV或ACV的基因治疗是目前肿瘤基因治疗领域中一种较有希望的治疗方法．反转录病毒和腺病毒介导的HSV－tk／NAS肿瘤基因治疗系统的建立,并运用该系统进行脑胶质瘤、垂体瘤、黑色素瘤的基因治疗离体细胞和动物试验,取得了显著的肿瘤杀伤作用,特别是腺病毒介导的脑胶质瘤基因治疗动物试验效果很显著,能够完全消除肿瘤．同时,还建立了反转录病毒以及腺病毒HSV－tk基因治疗的安全性检测体系,为基因治疗的临床试验提供了安全性保证．     

HSVTK gene therapy for carcinoembryonic antigen-producing human lung cancer cells

The long-term success of gene therapy for cancer relies heavily on the development of effective targeting systems. We investigate the possibility of targeted gene therapy using promoter of carcinoembryonic antigen (CEA) gene. By using luciferase reporter gene, we found that CEA promoter exhibit 16 times high activity in CEA-producing lung cancer cells, A549 than in nonproducing cells, Hela. We also constructed a recombinant expression plasmid pCEATK, in which CEA promoter drives the effector gene, thymidine kinase gene of Herpes Simplex Virus (HSVTK). A549 cells transfected with pCEATK became 865 times more sensitive to ganciclovir (GCV) than the control cells. However, Hela cells transfected with this plasmid remained resistant to GCV. These data indicate the potential for targeted gene therapy using the CEA promoter against CEA-producing tumor cells, such as lung cancer cells. Foundation item: Supported by the National Natural Science Foundation of China Natural Science Foundation of Hubei Province Biography: XIAO Geng-fu(1966-), male, phD graduate candidate, Lecturer.     

Progress in artificial control system for gene expression

Along with the increasingly wide application of transgenic techniques, new stricter criteria have been raised for controlling the expression of exogenous genes. For these demands, a series of artificial control systems for gene expression have been developed and testified in recent years, which can control exogenous genes expression in exact time and certain level by administration of a specific drug or hormone. The successful construction of these systems offers a practicable method to control precise expression of exogenous gene in organisms, and raises the feasibility of wide application of gene therapy.     

几种新型超分子配合物的合成和表征

制备3种新型超分子配合物并运用常规方法进行表征,讨论配体的特征骨架、空间配位方式和氢键作用对超分子形成的影响.以实验证实,从分子设计,通过分子自组装,是合成具有超分子结构配合物的有效途径.同时培养出化合物的高质量单晶,解析了晶体结构,分析和探讨了超分子配合物的组成-结构-性质之间的关系.     

纳米材料在基因治疗中的研究进展:从肿瘤治疗到新冠病毒疫苗

基因治疗作为一种精准有效的策略,可用于治疗包括癌症在内的多种疾病以及预防病毒性感染疾病．然而,基因治疗中使用的核酸药物自身不稳定性和大尺寸阻碍了它们的广泛应用．纳米材料因其免疫原性低、可控性好、易于进行表面修饰等特点,已被证明是基因治疗中最有前途的载体之一．特别是新型冠状病毒肺炎（COVID-19）疫情爆发以来,与传统疫苗不同的mRNA疫苗备受关注,而脂质纳米颗粒（LNP）作为该疫苗的递送系统发挥了至关重要的作用,展现了纳米材料巨大的应用前景．本文概述了基因治疗的主要类型;介绍了基于脂质的纳米颗粒、基于聚合物的纳米颗粒、无机纳米颗粒及新型纳米材料如碳点等核酸递送平台;强调了其在肿瘤治疗和COVID-19核酸疫苗中的最新研究进展;提出了纳米材料用于基因治疗的挑战与前景．      

Functional DNA Materials Controlled by Surrounding Conditions

1 Results Addressable, controllable, and switchable supramolecular devices can provide keys to regulate the structure and function of nanomaterials. From this viewpoint, oligonucleotides are promising supramolecular materials because their assembly is addressable and they can be programmed. The G-quadruplexes of the oligonucleotide possess at least two important aspects: functions in vivo and applications in vitro. In addition, it is demonstrated that the G-quadruplex is promising for nanomolecular mach...     

脂质体--基因载体

概述了基因载体的作用、类型和脂质体用于基因载体的发展历史,介绍了一类很有前途的基因载体．阳离子脂质体的组成结构和分类及其转基因的作用原理,讨论了影响阳离子脂质体／DNA复合物物理化学性质的参数及其目标复合物的制备方法．