基因治疗新药核酶的研究进展

对基因治疗新药核酶的结构、作用机制、临床和基础应用进行综述及展望。     

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。     

质粒DNA类基因治疗药物的研究现状及发展趋势

基因治疗是将外源基因导入人体以纠正基因缺陷的方法,通过转录和翻译实现对细胞基因表达的调控,从而合成特异性蛋白治疗相关疾病。质粒DNA是经基因工程改造过的环状DNA分子,其结构简单,具有自主复制能力,可以携带治疗基因导入人体细胞,被广泛用于基因治疗的研究中。目前已上市及正处于临床研究的基于质粒DNA的基因药物包括cambiogenplasmid、Tavo等。对遗传病、恶性肿瘤等适应证,质粒DNA的基因治疗比传统的治疗方案有明显优势。CRISPR-Cas9等相关的质粒基因编辑技术成为质粒DNA类基因治疗的一大研发趋势,基因枪、聚合物纳米载体等递送系统以透皮给药、静脉注射等方式为质粒DNA导入体内提供了更多可能。基于质粒DNA类的基因治疗已成为基因治疗领域的一种理想技术手段。     

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。     

AAV基因治疗药物生产车间工艺设计探讨及案例分析

以AAV基因治疗药物的生产工艺流程为基础,通过功能间进行划分,结合GMP和生物安全防护要求,提出合理的功能间布局和工艺流线,制定合适的人物流净化策略。结合实际设计案例,合理地规划功能区划及工艺流线,制定洁净分区和压差,提出相应的生物安全防护措施。     

DNA的应激性损伤

DNA损伤可激活一系列复杂的调节DNA修复以及激活细胞周期检验点的信号网络。Yaffe等最近指出，在经过DNA损伤类抗肿瘤药物处理后，p53缺陷的肿瘤细胞依赖一个可选择的、平行的信号途径来激活细胞周期检验点。     

Clinical potential of electroporation for gene therapy and DNA vaccine delivery

ABSTRACT

Introduction: Electroporation allows efficient delivery of DNA into cells and tissues, thereby improving the expression of therapeutic or immunogenic proteins that are encoded by plasmid DNA. This simple and versatile method holds a great potential and could address unmet medical needs such as the prevention or treatment of many cancers or infectious diseases.

Areas covered: This review explores the electroporation mechanism and the parameters affecting its efficacy. An analysis of past and current clinical trials focused on DNA electroporation is presented. The pathologies addressed, the protocol used, the treatment outcome and the tolerability are highlighted. In addition, several of the possible optimization strategies for improving patient compliance and therapeutic efficacy are discussed such as plasmid design, use of genetic adjuvants for DNA vaccines, choice of appropriate delivery site and electrodes as well as pulse parameters.

Expert opinion: The growing number of clinical trials and the results already available underline the strong potential of DNA electroporation which combines both safety and efficiency. Nevertheless, it remains critical to further increase fundamental knowledge to refine future strategies, to develop concerted and common DNA electroporation protocols and to continue exploring new electroporation-based therapeutic options.     

A theoretical study on interactions between mitoxantrone as an anticancer drug and DNA: application in drug design

This research is an effort to further understand the physicochemical interaction between the novel drug, mitoxantrone (MTX) and its biologic receptor, DNA. The ultimate goal is to design drugs that interact more with DNA. Understanding the physicochemical properties of the drug as well as the mechanism by which it interacts with DNA, it should ultimately allow the rational design of novel anti-cancer or anti-viral drugs. Molecular modelling on the complex formed between MTX and DNA presented that this complex was indeed fully capable of participating in the formation of a stable intercalation site. Furthermore, the molecular geometries of MTX and the DNA bases (adenine, guanine, cytosine and thymine) were optimized with the aid of the B3LYP/6-31G* method. The properties of the isolated intercalator and its stacking interactions with the adenine...thymine (AT) and guanine...cytosine (GC) nucleic acid base pairs were studied with the DFTB method (density functional tight-binding), an approximate version of the DFT method, that was extended to cover the London dispersion energy. The B3LYP/6-31G* stabilization energies of the intercalator...base pair complexes were found 10.06 kcal/mol and 21.64 kcal/mol for AT...MTX and GC...MTX, respectively. It was concluded that the dispersion energy and the electrostatic interaction contributed to the stability of the intercalator.DNA base pair complexes. The results concluded from the comparison of the DFTB method and the Hartree-fock method point out that these methods show close results and support each other.     

Recent developments in cationic lipid-mediated gene delivery and gene therapy

Gene therapy will revolutionize medicine, helping us to cure and prevent diseases at their core level. Until becoming a widespread reality, the problem of efficient gene transfer and expression (transfection) must be solved. Cationic lipids represent a safer alternative than viral vectors, which, although more efficient, have the drawback of immunogenicity and propagation risks. Additionally, cationic lipids and cationic liposomes allow the delivery of larger plasmids and may be GMP manufactured and stored in bulk quantities. However, their specific transfection efficiency must be improved in order to reach the performance of biological vectors. In recent years, new structures have been released and tested, with designs adapted to recent findings in lipid-mediated transfection mechanisms. Another trend is the increased use of natural, biodegradable, building blocks in the backbone of these compounds. Here we review the very recent developments in the field of cationic lipids, both from industry and academia. Physicochemical characteristics, insights of transfection mechanisms, as well as therapeutic applications are also presented. Finally, some future prospects and trends are proposed.     

RNA组合干扰技术及其在肿瘤基因治疗中的应用

RNA干扰(RNAi)为基因治疗提供了一个全新的思路。但单一的RNAi治疗只针对单个基因,因而最终无法治愈多基因变异引起的肿瘤。为了克服RNAi单基因治疗的不足,研究人员提出了RNA组合干扰(combinatorial RNA interference,coRNAi)的治疗策略。coRNAi通过联合应用针对单个或多个靶基因的RNAi诱发物以及其他RNA或蛋白沉默因子实现沉默靶基因、抑制肿瘤生长和促进肿瘤细胞凋亡。本文重点介绍了coRNAi的主要联合方式以及在肿瘤基因治疗中的研究进展。     

New strategies for cancer gene therapy: Progress and opportunities

1. To date, cancer persists as one of the most devastating diseases worldwide. Problems such as metastasis and tumour resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of existing clinical treatments.
2. To address these problems, cancer gene therapy has been developing over the past two decades, specifically designed to deliver therapeutic genes to treat cancers using vector systems. So far, a number of genes and delivery vehicles have been evaluated and significant progress has been made with several gene therapy modalities in clinical trials. However, the lack of an ideal gene delivery system remains a major obstacle for the successful translation of regimen to the clinic.
3. Recent understanding of hypoxic and necrotic regions within solid tumours and rapid development of recombinant DNA technology have reignited the idea of using anaerobic bacteria as novel gene delivery systems. These bacterial vectors have unique advantages over other delivery systems and are likely to become the vector of choice for cancer gene therapy in the near future.
4. Meanwhile, complicated tumour pathophysiology and associated metastasis make it hard to rely on a single therapeutic modality for complete tumour eradication. Therefore, the combination of cancer gene therapy with other conventional treatments has become paramount.
5. The present review introduces important cancer gene therapy strategies and major vector systems that have been studied so far with an emphasis on bacteria-mediated cancer gene therapy. In addition, exemplary combined therapies are briefly reviewed.     

Reducing the immunostimulatory activity of CpG-containing plasmid DNA vectors for non-viral gene therapy

The mammalian innate immune system has the ability to recognise and direct a response against incoming foreign DNA. The primary signal that triggers this response is unmethylated CpG motifs present in the DNA sequence of various disease-causing pathogens. These motifs are rare in vertebrate DNA, but abundant in bacterial and some viral DNAs. Because gene therapy generally involves the delivery of DNA from either plasmids of bacterial origin or recombinant viruses, an acute inflammatory response of variable severity inevitably results. The response is most serious for non-viral gene delivery vectors composed of cationic lipid–DNA complexes, producing adverse effects at lower doses and lethality at higher doses of complex. This review examines the role of immunostimulatory CpG motifs in the acute inflammatory response to non-viral gene therapy vectors. Strategies to neutralise or eliminate CpG motifs within plasmid DNA vectors, and the existing limitations of CpG reduction on improving the safety profile of non-viral vectors, will be discussed.     

Advances in skin gene therapy

Specific anatomical and biological properties make the skin a very interesting target organ for gene therapy approaches. Different cell types of the epidermis, such as keratinocytes, melanocytes, or dendritic cells, can be genetically modified to treat a broad spectrum of diseases, including genetically inherited skin disorders, tumour diseases, metabolic disorders and infectious diseases. The easy accessibility of skin suggests that different methods for gene delivery can be pursued, depending on the desired application. The approach used to deliver DNA to the skin will influence not only the efficiency of DNA delivery, but also the level and duration of transgene expression. Furthermore, the desired biological effect will also influence the decision of which gene transfer method is the best choice. Among the current challenges of cutaneous gene therapy are: optimising the efficiency of direct in vivo gene delivery; targeting specific epidermal cells, including keratinocyte stem cells; achieving sustained gene expression and regulating gene expression in vivo. This review summarises recent advances in the field of skin gene therapy and evaluates possible strategies to overcome obstacles and achieve successful clinical applications of skin gene therapy.     

靶向诱导DNA甲基化与肿瘤治疗研究进展

DNA甲基化是表观遗传调控的重要方式之一,它能在转录水平上抑制相关基因的表达,从而引起基因沉默。近年来的研究表明,DNA的甲基化不仅在生物体内自然发生,而且可通过特异的RNA、DNA等靶向诱导产生。肿瘤的细胞中存在癌基因的异常低甲基化,而且这种异常被认为是肿瘤的发病机制之一。因此,通过靶向诱导癌基因甲基化来抑制癌基因的表达、抑制肿瘤生长的方法,为肿瘤治疗提供了一种新的思路。本文就靶向诱导DNA甲基化与肿瘤治疗的临床前研究进展作一简述。     

卵巢癌基因治疗的新进展

卵巢癌是恶性度最高的妇科肿瘤之一，由于手术和化疗的不尽人意，基因治疗是当今肿瘤研究的热点。卵巢癌的基因治疗载体主要分为病毒载体系统和非病毒载体系统。其中，腺病毒载体转染效率较高，是目前基因治疗的主要手段和最有前途的载体。卵巢癌的基因治疗策略有分子化学治疗，基因替代和免疫系统的基因调控。由于这一领域的迅速发展，卵巢癌的基因治疗有着光明的前景。