腺相关病毒介导锌指核酸酶体外切除HIV-1基因组研究

人类免疫缺陷病毒1型(Human Immunodeficiency Virus type 1, HIV-1)能够整合到宿主细胞基因组并通过基因沉默的方式逃避高效抗逆转录病毒疗法(Highly Active Antietroviral Therapy, HAART),而高效特异性的基因编辑工具的出现，有望成功切除HIV-1前病毒基因组。重组腺相关病毒(AAV)是基因治疗的重要载体系统，为了获得能够高效感染T细胞的基因编辑工具递送系统，本研究通过比较过表达增强绿色荧光蛋白的血清型2、血清型6、血清型DJ的重组腺相关病毒，筛选出T细胞系的偏好血清型为6型。通过比较CMV启动子、SFFV启动子，筛选出T细胞系的偏好启动子为SFFV启动子。最后，我们通过制备过表达靶向HIV-1长末端重复序列(Long Terminal Region, LTR)的锌指核酸酶(Zinc Finger Nuclease, ZFN)的AAV6递送载体系统，感染模拟HIV-1阳性表达的细胞系Ya, T7E1检测证实HIV-1前病毒基因组被特异性切除，流式细胞仪检测证实切除效率约为17.1%,测序分析进一步证实靶向HIV-...     

光控超小型CRISPR/Cas经AAV载体递送实现对人基因组的高效编辑

CRISRP/Cas系统是目前广为使用的基因编辑工具，而腺相关病毒(AAV)也是目前最安全有效的基因治疗递送载体。为了实现AAV载体介导的CRISRP/Cas基因编辑，各种可装载于AAV载体的基因编辑工具也先后被报道。尽管如此，AAV载体介导的时空特异性基因编辑仍较难实现。为此我们结合光遗传学，将蓝光诱导表达系统、超小型CRISPR/Un1Cas12f1基因编辑系统以及AAV载体递送系统进行有机整合，开发出光控诱导表达的超小型基因编辑系统(AAV-PA-Cas12f1),并将其通过AAV递送至人源细胞HEK293T中。在无光条件下其表达背景极低，而经过蓝光诱导实现了对人基因组的高效基因编辑：以Intergene2为例，利用PCR产物-TA克隆结合单克隆测序分析得到该系统的实际编辑效率高达56%,编辑类型为碱基缺失或兼具碱基缺失与插入。此工具的开发为实现体内精准基因编辑奠定了基础，为解析更多生物学机制及探索疾病治疗策略提供了技术平台。     

单基因遗传疾病的基因疗法

 基因疗法是全球突破性技术之一，在单基因遗传疾病治疗中已取得突破性进展。阐述了不同的基因疗法策略、载体和基因编辑技术的特点，综述了脊髓性肌萎缩症、Leber先天性黑蒙2型、血友病、β-地中海贫血的发病机理、临床表现、基因疗法的开发进程以及临床试验情况。目前，上述4种单基因疾病的9种基因疗法已分别取得美国食品药品监督管理局突破性疗法资格、欧洲药品管理局的优先药物资格或者已经批准上市。基因疗法的研究还面临着许多挑战，但随着科学研究的深入和科学技术的不断发展，将有更多的患者获得治疗。     

基因治疗中目的基因的导入方法

基因治疗正从实验室研究走向临床应用，它给医学界引入了一种全新的概念，基因治疗主要是利用病毒介导的基因转移。文中介绍了目前已应用和将要应用于人类基因治疗的逆转录病毒载体、腺病毒载体、腺相关病毒载体、疱疹病毒载体等的构建方法，优缺点和适于治疗的疾病。扼要地介绍了目的基因导入的理化方法。     

病毒载体滴度对基因转移效率的影响研究

用改良的经典方法测定包装ＬａｃＺ基因的腺病毒和逆转录病毒载体的病毒滴度，以及用新建的简便方法测定逆转录病毒载体的病毒滴度，应用此两种病毒载体介导ＬａｃＸＺ基因分别导入１５种人或小鼠靶细胞，并以不同滴度的病毒上清液转染靶细胞Ｋ５６２，探讨病毒滴度对基因转移效率的影响，实验结果表明，病毒滴度与转染效率呈正相关，逆转录病毒滴度由１０＾６ＣＦＵ／ｍＬ。     

罕见病诊断的相关技术及发展

 罕见病大多数是遗传性疾病,其发病率低,种类繁多且表型复杂多样,导致临床上难以进行及时和准确的诊断。随着分子遗传学、分子诊断技术、基因测序技术及组学技术的进步,罕见病诊断取得了重大发展。在传统基因检测技术基础上,二代测序技术迅速发展并广泛应用于罕见病的诊断和研究中,三代测序技术也展示出潜在应用价值。虽然传统的酶学检测技术仍占有重要地位,但已不能满足罕见病诊断的需求;蛋白质组学、代谢组学的崛起,使多种罕见病的准确诊断成为可能。同时结合分子影像技术和生物信息技术,计算机辅助诊断也展现出了广泛的应用前景。     

人类基因治疗述评（二）

3　关于非病毒载体的设计策略在基因治疗的研究中,关于载体及其改造的研究,关于基因调控的研究,关于非病毒载体的研究均特别引人注目。这里略谈非病毒载体的设计策略。基因转移的非病毒技术近年来取得了迅速的进步。尽管现在多用病毒来转染哺乳动物细胞,但存在一些目前的?..     

猕猴B病毒妒和gC合成基因重组杆状病毒质粒的构建

目的构建猕猴B病毒gB和gc合成基因的杆状病毒重组质粒（rBacmid）。方法根据本室已经合成的猕猴B病毒gB基因和gc基因,利用BamHI和XhoI特异性酶切,将其亚克隆于昆虫杆状病毒的pFastBacHTA转座载体。将该阳性转座载体转化到DHlobac感受态细胞,从中筛选出阳性重组细菌,并提取重组质粒。结果获得了重组质粒bacmid—gB和bacmid—gC。结论该研究为猕猴B病毒gB和gc蛋白在杆状病毒系统内的表达奠定了基础。     

基因疗法首次降伏HIV

<正>一项临床试验日前表明,一种基因编码技术对于人类而言是安全且有效的。这是研究人员第一次使用一种名为锌指核酸酶(ZFN)的酶瞄准并破坏了12名艾滋病病毒(HIV)携带者免疫细胞中的一种基因,从而增强了他们抵抗病毒的能力。该项研究成果发表在3月6日出版的《新英格兰医学杂志》上。美国加利福尼亚州杜瓦迪市希望之城国家医学中心贝克曼研究所分子生物学家John Rossi说:"这是HIV基因疗法的第一个重大进步,因为它证明了‘柏林病人’Timothy Brown如何远离HIV。"     

小鼠Sirt3基因过表达慢病毒载体的构建及其表达鉴定

构建小鼠Sirt3基因过表达慢病毒载体,检测人神经母细胞瘤细胞(SK-N-SH细胞)感染Sirt3基因过表达慢病毒后Sirt3 mRNA和蛋白的表达,为在细胞和动物水平研究Sirt3的作用提供新的途径和工具。利用Genebank检索的小鼠Sirt3基因序列,人工合成法合成小鼠Sirt3基因的c DNA片段,将其克隆至慢病毒载体p CDH-CMV-MCS-EF1-cop GFP中,构建慢病毒表达质粒。进行酶切及测序验证后,将慢病毒表达质粒连同辅助包装原件载体质粒共同转染入293T细胞,收集上清,测定病毒滴度。用Sirt3基因过表达慢病毒感染SK-N-SH细胞,即为Sirt3过表达组(Sirt3);用空载体慢病毒感染的SK-N-SH细胞为空载体组(GFP);未感染病毒的SK-N-SH细胞为对照组(CON)。收集细胞后,用Real-time PCR法检测各组细胞Sirt3 mRNA的水平;用Western blotting法检测各组细胞Sirt3蛋白的表达。Sirt3基因过表达慢病毒载体构建成功,病毒滴度为1.0×10~9TU/m L。Sirt3组SK-N-SH细胞的Sirt3 mRNA和Sirt3蛋白水平均显著高于GFP组和CON组(P0.01)。成功构建的Sirt3基因过表达慢病毒载体具备高效感染力,能在SK-N-SH细胞高效过表达Sirt3,本研究结果为今后进一步在神经细胞和模型动物中研究Sirt3的作用提供了新的途径和工具。     

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.     

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.     

Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity

A common viral immune evasion strategy involves mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of anti-vector immunity. Recombinant, replication-incompetent adenovirus serotype 5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens have proved highly immunogenic in preclinical studies but will probably be limited by the high prevalence of pre-existing anti-Ad5 immunity in human populations, particularly in the developing world. Here we show that rAd5 vectors can be engineered to circumvent anti-Ad5 immunity. We constructed novel chimaeric rAd5 vectors in which the seven short hypervariable regions (HVRs) on the surface of the Ad5 hexon protein were replaced with the corresponding HVRs from the rare adenovirus serotype Ad48. These HVR-chimaeric rAd5 vectors were produced at high titres and were stable through serial passages in vitro. HVR-chimaeric rAd5 vectors expressing simian immunodeficiency virus Gag proved comparably immunogenic to parental rAd5 vectors in naive mice and rhesus monkeys. In the presence of high levels of pre-existing anti-Ad5 immunity, the immunogenicity of HVR-chimaeric rAd5 vectors was not detectably suppressed, whereas the immunogenicity of parental rAd5 vectors was abrogated. These data demonstrate that functionally relevant Ad5-specific neutralizing antibodies are focused on epitopes located within the hexon HVRs. Moreover, these studies show that recombinant viral vectors can be engineered to circumvent pre-existing anti-vector immunity by removing key neutralizing epitopes on the surface of viral capsid proteins. Such chimaeric viral vectors may have important practical implications for vaccination and gene therapy.     

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．      

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.     

Red/ET同源重组系统介导的质粒载体快速构建

Red/ET重组技术是近年来建立的一种基于高效率体内同源重组的新型遗传工程技术,该技术简便、快速.首先,通过一步融合的方法将链霉素抗性基因和氯霉素抗性基因同时克隆至pUC19上,从而构建出含有正反选择标记的载体pRC;其次,用安普霉素抗性基因或者氯霉素抗性基因替换了常用表达载体pET-28b上的卡那霉素抗性基因,改变了其抗性选择标记,得到的表达载体pMT和pCT可以更广泛地应用于不同宿主中;最后,还将启动和终止表达的区域定点插入pACYC184载体中,使其成为可以独立表达蛋白的表达载体p184.本研究所得到的载体为大肠杆菌宿主菌中的基因克隆和蛋白表达提供了基础.