Basic Principles and Clinical Applications of CRISPR-Based Genome Editing

Advances in sequencing technologies have facilitated the discovery of previously unknown genetic variants in both inherited and acquired disorders, and tools to correct these pathogenic variants are rapidly evolving. Since the first introduction of CRISPR-Cas9 in 2012, the field of CRISPR-based genome editing has progressed immensely, giving hope to many patients suffering from genetic disorders that lack effective treatment. In this review, we will examine the basic principles of CRISPR-based genome editing, explain the mechanisms of new genome editors, including base editors and prime editors, and evaluate the therapeutic possibilities of CRISPR-based genome editing by focusing on recently published clinical trials and animal studies. Although efficacy and safety issues remain a large concern, we cannot deny that CRISPR-based genome editing will soon be prevalent in clinical practice.     

Cellular and Tissue Selectivity of AAV Serotypes for Gene Delivery to Chondrocytes and Cartilage

Background: Despite several studies on the effect of adeno-associated virus (AAV)-based therapeutics on osteoarthritis (OA), information on the transduction efficiency and applicable profiles of different AAV serotypes to chondrocytes in hard cartilage tissue is still limited. Moreover, the recent discovery of additional AAV serotypes makes it necessary to screen for more suitable AAV serotypes for specific tissues. Here, we compared the transduction efficiencies of 14 conventional AAV serotypes in human chondrocytes, mouse OA models, and human cartilage explants obtained from OA patients.Methods: To compare the transduction efficiency of individual AAV serotypes, green fluorescent protein (GFP) expression was detected by fluorescence microscopy or western blotting. Likewise, to compare the transduction efficiencies of individual AAV serotypes in cartilage tissues, GFP expression was determined using fluorescence microscopy or immunohistochemistry, and GFP-positive cells were counted.Results: Only AAV2, 5, 6, and 6.2 exhibited substantial transduction efficiencies in both normal and OA chondrocytes. All AAV serotypes except AAV6 and rh43 could effectively transduce human bone marrow mesenchymal stem cells. In human and mouse OA cartilage tissues, AAV2, AAV5, AAV6.2, AAV8, and AAV rh39 showed excellent tissue specificity based on transduction efficiency. These results indicate the differences in transduction efficiencies of AAV serotypes between cellular and tissue models.Conclusions: Our findings indicate that AAV2 and AAV6.2 may be the best choices for AAV-mediated gene delivery into intra-articular cartilage tissue. These AAV vectors hold the potential to be of use in clinical applications to prevent OA progression if appropriate therapeutic genes are inserted into the vector.     

CRISPR-Cas9技术在人类疾病治疗中的潜在应用

CRISPR-Cas9的发明为生物学领域带来一场新的技术革命。 CRISPR-Cas系统是一种存在于多种细菌和古菌内针对外源遗传物质侵袭的获得性免疫系统,由规律性重复短回文序列簇及其相关蛋白组成。近年该系统被发展成一种由CRISPR相关RNA介导的Cas9核酸内切酶靶向基因编辑技术,在科研、医学及生物技术等多个领域上被广泛应用。 CRISPR-Cas9技术多功能及可编程特点,令其在动物模型制备、物种改良等方面取得重要应用。同时在遗传性疾病、恶性肿瘤、感染性疾病等许多疾病的研究、治疗及预防上均显示出巨大应用潜力。     

壳聚糖基因纳米粒子的细胞摄入和体外毒性研究

用分子量为50kDa和400kDa的壳聚糖分别和[α-32P]dATP标记的质粒DNA,在不同的N/P比下,通过复凝聚方法形成基因纳米粒子。对形成的壳聚糖基因纳米粒子(Chitosan gene nanoparticle,CGN)进行表征,评价CGN体外细胞毒性,研究两种壳聚糖形成的基因纳米粒子被A10和K562细胞摄入的量和速度。结果表明:(1)随着壳聚糖分子量和N/P比的增大,形成的基因纳米粒子更易于进入细胞,同时显示纳米粒子的zeta电位与细胞摄入量之间存在关联性;(2)壳聚糖基因纳米粒子的毒性远远小于商品化的细胞转染试剂Lipofectamine2000。     

艾滋病的基因治疗策略及慢病毒载体

高活性的抗病毒治疗可以显著地降低艾滋病患者血浆中的HIV病毒载量,但对潜伏的病毒库无效。对HIV的基因治疗包括诱导HIV潜伏感染的休止的CD4 T记忆细胞增生,使潜伏的HIV激活进入复制循环,结合药物治疗和激活潜伏的HIV基因表达但并不诱导细胞增生,而是通过载体携带的基因使细胞凋亡,以清除HIV潜伏感染的细胞,利用载体携带目的基因治疗脑中的病毒。     

纳米生物技术基因治疗载体研究进展

基因治疗已在治疗多种人类重大疾病如遗传病、肿瘤等方面显示出良好的应用前景,但也面临着巨大的挑战,其中之一就是需要安全、高效、靶向的载体系统。纳米生物材料,如脂质体、聚丙交酯-乙交酯(PLGA),聚乳酸(PLA)等,由于具有良好的生物安全性、可方便有效地实现基因靶向性及高效表达和缓释,成为制备高效、靶向的基因治疗载体系统的良好介质,日益在基因治疗载体系统中受到广泛重视。本文综述了目前在基因治疗领域中常用的纳米载体的生物学特性,以及它们的最新研究进展。     

血友病的基因治疗

血友病是基因治疗临床实验最早获得成功的遗传性疾病.血友病不仅是单基因疾病治疗的最佳病种,因其清楚的遗传背景、完备的动物模型以及客观方便的观察指标,也成为检验基因治疗系统最为可信的模型.本文综述血友病基因治疗的代表性文献,对基因治疗血友病的进展、面临的困难以及可能的解决途径及其前景作一简要综述.     

Gene therapy to create biological pacemakers

Old age and a variety of cardiovascular disorders may disrupt normal sinus node function. Currently, this is successfully treated with electronic pacemakers, which, however, leave room for improvement. During the past decade, different strategies to initiate pacemaker function by gene therapy were developed. In the search for a biological pacemaker, various approaches were explored, including β₂-adrenergic receptor overexpression, down regulation of the inward rectifier current, and overexpression of the pacemaker current. The most recent advances include overexpression of bioengineered ion channels and genetically modified stem cells. This review considers the strengths and the weaknesses of the different approaches and discusses some of the different viral vectors currently used.     

脊柱融合的基因治疗

稳定的脊柱融合与相邻椎骨的融合有关，经典的脊柱融合手术采用剥离椎骨，自体髂骨植骨融合术，虽然应用自体髂骨植骨被认为是植骨融合的金标准，但是它常伴有并发症出现，基因治疗被认为是促进脊柱关节固定融合的新途径，研究表明在动物模型中应用腺病毒，脂质体搭载人骨形成蛋白（hBMP)基因族能促进脊柱融合，有关在动物模型中通过基因转染促进脊柱融合的研究，本文对此作简要介绍，弄清楚骨形成中基因的表达和开发出新的基因转染载体是目前研究的重点，相信在不远的将来，基因治疗脊柱融合能在临床中得到应用。     

GAD抗原肽抗体对IDDM糖尿病基因治疗研究

目的　探讨GAD抗原肽抗体诱导特异性免疫耐受对IDDM糖尿病的基因治疗。方法　构建Ig MBAE、GAD Ig MBAE融合蛋白表达载体 ,制备产重组病毒的病毒包装细胞 ,经逆转录病毒载体介导基因转染B细胞 ,对非胖型糖尿病小鼠 (NOD)进行基因治疗 ,以血糖指标和病理组织学分析评价疗效。结果　 1.酶切鉴定证实Ig MBAE、GAD Ig MBAE表达载体构建正确。 2 .RT PCR可检测到转染 4 8hGAD Ig B细胞中的GAD Ig融合基因表达 ;Westernblot和双夹心ELISA可检测到转染细胞胞浆和细胞上清中的GAD Ig融合蛋白 ;3.在治疗第 36周 ,GAD Ig B细胞治疗组的糖尿病发病率由B细胞和Ig B细胞组的10 0 %降低至 5 5 .5 % ,胰腺组织病理分析提示 ,GAD Ig B细胞治疗组的胰岛组织形态接近正常 ,胰岛组织中淋巴单核细胞的浸润得到有效控制。结论　GAD抗原肽抗体通过逆转录病毒介导的基因治疗 ,可有效地延迟NOD小鼠的糖尿病发病及明显降低发病率。     

Ultra-Sensitive Droplet Digital PCR for the Assessment of Microchimerism in Cellular Therapies

Current techniques to assess chimerism after hematopoietic stem cell transplantation (HSCT) are limited in both sensitivity and precision. These drawbacks are problematic in the context of cellular therapies that frequently result in microchimerism (donor chimerism <1%). We have developed a highly sensitive droplet digital PCR (ddPCR) assay using commercially available regents with good performance throughout the range of clinically relevant chimerism measurements, including microchimerism. We tested the assay using spiked samples of known donor-recipient ratios and in clinical samples from HSCT recipients and patients enrolled on clinical trials of microtransplantation and third-party virus-specific T cells (VSTs). The levels of detection and quantification of the assay were .008% and .023%, with high levels of precision with samples of DNA content ranging from 1 to 300?ng DNA. From the panel of 29 insertion-deletion probes multiple informative markers were found for each of 43 HSCT donor-recipient pairs. In the case of third-party cellular therapies in which there were 3 DNA contributors (recipient, HSCT donor, and T-cell donor), a marker to detect the cellular product in a background of recipient and donor cells was available for 11 of 12 cases (92%). Chimerism by ddPCR was able to quantify chimerism in HSCT recipients and comparison against standard STR analysis in 8 HSCT patients demonstrated similar results, with the advantage of fast turnaround time. Persistence of donor microchimerism in patients undergoing microtransplantation for acute myeloid leukemia was detectable for up to 57 days in peripheral blood and bone marrow. The presence of microtransplant product DNA in bone marrow T cells after cell sorting was seen in the 1 patient tested. In patients receiving third-party VSTs for treatment of refractory viral infections, VST donor DNA was detected at low levels in 7 of 9 cases. ddPCR offers advantages over currently available methods for assessment of chimerism in standard HSCT and cellular therapies.     

鼻咽癌基因治疗研究的新进展

目的：总结鼻咽癌基因治疗研究进展,为临床治疗及基础研究提供参考。方法：在万方、中国知网、pubmed数据库检索近年关于鼻咽癌基因治疗的研究资料,并进行综合分析。结果：鼻咽癌基因治疗在基础研究方面取得满意的效果,在临床应用中副作用少、医从性好。具体方法有免疫基因治疗,自杀基因治疗,基因替代治疗,基因沉默治疗和反义基因治疗。结论：基因治疗将成为鼻咽癌治疗的重要手段。     

高血压基因治疗的研究进展

高血压是导致全世界成人死亡和瘫痪的一个重要原因，它显著增加心血管疾病的危险性，包括脑卒中、冠心病、心力衰竭、心律失常、心肌病等。在动物试验中获得的数据显示基因治疗高血压是可行的，另一方面传统的降压药物自身也存在一些缺点，基因治疗高血压已成为研究治疗高血压的基本方向。本文概述了在治疗高血压过程中基因治疗的作用以及当前基因治疗的研究难点，并分析了高血压的基因治疗发展前景。     

Gene Therapeutic Approaches to Induction and Maintenance of Tolerance

Tolerance induction would be an ideal way to treat autoimmune diseases, especially if achievable in primed individuals. Moreover, specific tolerance would preclude the need for immunosuppressive treatment with its side effects. In this review, we will revisit the historical concepts of tolerance, and introduce a novel approach to tolerance via gene therapy. Our model system is based on the tolerogenicity of IgG carriers and B-cell antigen presentation. Results with this model demonstrate that it is simple and effective even in primed recipients, and has proven efficacy in three autoimmune models. We discuss the mechanisms of tolerance with fusion IgG's and analyze how our model of gene therapy approached can be utilized to fit in the future treatment of autoimmune conditions.     

树枝状高分子在生物医学领域的应用研究进展

树枝状大分子 (dendrim ers)是近年来出现的一类新型合成高分子 ,具有高度支化、结构规整、单分散等特性。这些特点使其在许多领域有潜在的重要应用价值 ,成为相关领域的研究热点。本文介绍了树枝状大分子的基本结构和合成路线 ,并对近年来国际上关于树枝状大分子在药物运载、DNA传递、特殊造影剂、肿瘤治疗新技术等生物医学领域的研究及应用探索进行了综述。     

Teaching Cellular Engineering

Cellular engineering is one of the fastest growing subdisciplines in the field of Biomedical Engineering. It involves the application of engineering analysis to understand and control cellular behavior, with the ultimate objective of developing novel therapeutic or diagnostic approaches for the clinic or harnessing cellular function for commercial applications. Well-educated students in this area need strong foundational knowledge in engineering science, chemistry, and cell and molecular biology. In undergraduate curricula, the challenge is to include essential engineering skills plus appropriate levels of training in chemistry and biology while satisfying accreditation-mandated breadth in engineering training. At the graduate level, educators must accommodate students with diverse backgrounds and provide them with both a state-of-the-art understanding of the life sciences and the most advanced engineering skills. Engineering curricular content should include mechanics and materials, physical chemistry, transport phenomena, and control theory. Training from faculty with appointments and research programs in the life sciences is generally recommended, and additional life science content should also be integrated within the engineering curriculum. A capstone course in cellular engineering that includes opportunities for students to have hands-on experiences with state-of-the-art laboratory techniques is highly recommended.     

Reprogrammed quiescent B cells provide an effective cellular therapy against chronic experimental autoimmune encephalomyelitis

Activated B cells can regulate immunity and have been envisaged as a potential cell-based therapy for treating autoimmune diseases. However, activated human B cells can also propagate immune responses, and the effects resulting from their infusion into patients cannot be predicted. This led us to consider resting B cells, which in contrast are poorly immunogenic, as an alternative cellular platform for the suppression of unwanted immunity. Here, we report that resting B cells can be directly engineered with lentiviral vectors to express antigens in a remarkably simple, rapid, and effective way. Notably, this neither required nor induced activation of the B cells. With this approach we were able to produce reprogrammed resting B cells that inhibited antigen-specific CD4(+) T cells, CD8(+) T cells, and B cells upon adoptive transfer in mice. Furthermore, resting B cells engineered to ectopically express myelin oligodendrocyte glycoprotein antigen protected recipient mice from severe disability and demyelination in EAE, and even induced complete remission from disease in mice lacking functional natural Tregs, which otherwise developed chronic paralysis. In conclusion, our study introduces reprogrammed quiescent B cells as a novel tool for suppressing undesirable immunity.     

脉冲电场生物学效应及医学应用

脉冲电场能够产生许多不同于一般电场的生物学效应,尤其是纳秒脉冲对细胞的作用更为特殊。本文主要就不同的脉冲电场对细胞膜,胞内基质,细胞增殖等产生的生物学效应作相应的介绍。并在此基础之上分析多种生物效应在肿瘤治疗、基因治疗、以及药物输送等方面的研究现状。