慢病毒载体定量检测方法的研究进展

免疫疗法正在发展成为一种可用于多种恶性肿瘤的有效且侵入性较小的治疗方法。慢病毒载体(Lentiviral vectors,LVs)因其能够稳定地整合相对较大的外源DNA,并且有效地转导分裂细胞和非分裂细胞,已在免疫治疗中显现出巨大潜力。临床应用对慢病毒载体具有较高的质量要求,需要对最终产品进行严格的质量控制以保证其纯度、效力和安全性。其中慢病毒载体的定量检测是产品研发和质控的关键环节之一。文中总结了LVs定量检测的现有方法,包括流式细胞术(Fluorescence activated cell sorter,FACS)、P24酶联免疫法(P24 enzyme-linked immunosorbent assay,P24 ELISA)、实时荧光定量PCR方法 (Real-time fluorescence quantitative polymerase chain reaction,RT-q PCR)、纳米粒子跟踪分析(Nanoparticle tracking analysis,NTA)、可调电阻式脉冲传感(Tunable resistive pulse sensing,TRPS)和病毒计数仪(Virus counter,VC),并对其优缺点进行比较,对发展新的检测方法和存在的挑战进行了展望。     

Advances in novel influenza vaccines: a patent review

The threat of a major human influenza pandemic such as the avian H5N1 or the 2009 new H1N1 has emphasized the need for effective prevention strategies to combat these pathogens. Although egg based influenza vaccines have been well established for a long time, it remains an ongoing public health need to develop alternative production methods that ensures improved safety, efficacy, and ease of administration compared with conventional influenza vaccines. This article is intended to cover some of the recent advances and related patents on the development of influenza vaccines including live attenuated, cell based, genomic and synthetic peptide vaccines.     

Surface-engineering of lentiviral vectors

Vectors derived from retroviridae offer particularly flexible properties in gene transfer applications given the numerous possible associations of various viral surface glycoproteins (determining cell tropism) with different types of retroviral cores (determining genome replication and integration). Lentiviral vectors should be preferred gene delivery vehicles over vectors derived from onco-retroviruses such as murine leukemia viruses (MLVs) that cannot transduce non-proliferating target cells. Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors, their interaction with the host immune system and their host range. There are however important gene transfer restrictions to some non-proliferative tissues or cell types and recent studies have shown that progenitor hematopoietic stem cells in G(0), non-activated primary blood lymphocytes or monocytes were not transducible by lentiviral vectors. Moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer applications in vivo. Several innovative approaches have been explored to overcome such problems that have given rise to novel concepts in the field and have provided promising results in preliminary evaluations in vivo. Here we review the different approaches explored to upgrade lentiviral vectors, aiming at developing vectors suitable for in vivo gene delivery.     

Production and purification of lentiviral vectors

Lentiviral vectors offer unique versatility and robustness as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the transgene both in vitro and in vivo. This protocol describes how lentiviral vectors can be produced, purified and titrated. High titer suspensions can be routinely prepared with relative ease: a low-titer (10(6) viral particles/ml) unpurified preparation can be obtained 3 d after transfecting cells with lentiviral vector and packaging plasmids; a high-titer (10(9) viral particles/ml) purified preparation requires 2 more days.     

Advances in biomedical sensor technology: A review of the 1985 patent literature

The 1985 patent literature pertaining to biomedical sensor technology is reviewed and an assessment made of the types of devices that are most likely to reach commercialisation. Novel systems that are described comprise electrochemical, optical, thermal and other transducers for monitoring key components of clinical samples. These include blood gases and electrolytes, metabolites, enzymes, proteins, antibodies and antigens.     

Recent advances in lentiviral vectors for gene therapy

Lentiviral vectors (LVs),derived from human immunodeficiency virus,are powerful tools for modifying the genes of eukaryotic cells such as hematopoietic stem cel...     

Efficient transgenesis in farm animals by lentiviral vectors

Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ-line. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.     

Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

Hepatocyte-specific gene expression from integrated lentiviral vectors

BACKGROUND: For many applications, efficient gene therapy will require long-term, organ-specific therapeutic gene expression. Lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Many experimental vectors express transgenes under the control of the cytomegalovirus (CMV) immediate-early gene promoter. Although this promoter directs strong gene expression in vitro, it may be shut off rapidly in vivo. This study explores the potential of HIV-1-based vectors to transduce hepatocytes and compares gene expression from different promoters in integrated vectors. METHODS: HIV-1-based vector plasmids expressing the green fluorescent protein (GFP) under the control of the CMV promoter, the alpha-1 antitrypsin gene promoter or promoters derived from the hepatitis B virus (HBV) genome were used to compare expression in transfected and transduced cell lines. RESULTS: Hepatocyte cell lines differed strikingly in their transfectability. Transduction with replication-deficient HIV-1-based vector particles incorporating the different promoter elements was uniformly effective in hepatocyte and non-hepatocyte lines. However, in hepatocytes, only the CMV, alpha-1 antitrypsin and HBV core but not HBV surface promoters were able to produce GFP expression. Addition of the HBV enhancer 2 element improved the transducing ability of the HBV surface promoter and suppressed expression in non-hepatocytes increasing specificity for hepatocytes. CONCLUSIONS: Integrated lentiviral vectors can be used to direct transgene expression in liver cells both promiscuously and specifically. Promoters derived from the alpha-1 antitrypsin gene or HBV are alternatives to the CMV promoter. Inclusion of the HBV enhancer 2 permits strong liver-specific gene expression in vitro.     

Reduced mobilization of Rev-responsive element-deficient lentiviral vectors

Infection of cells transduced with a lentiviral vector by human immunodeficiency virus (HIV) could lead to packaging of the lentiviral vector RNA into HIV particles and unintended transfer of the vector. To prevent this, the Rev-responsive element (RRE) of an HIV-1 vector was functionally replaced by a heterologous RNA element (MS2). Providing Rev fused to an MS2 binding protein allowed efficient vector production. Mobilization of the vector from infected target cells was below the level of detection and at least 10(3)- to 10(4)-fold lower than for the RRE-containing vector. Thus, RRE-deficient lentiviral vectors provide a novel approach to reduce the risk of vector mobilization.     

Effective gene therapy with nonintegrating lentiviral vectors

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.     

Tet system in the brain: Transgenic rats and lentiviral vectors approach

Local and regulated expression of exogenous genes in the central nervous system is one of the major challenges of modern neuroscience. We have approached this issue by applying the inducible tetracycline system to regulate the expression of EGFP reporter gene in double transgenic rats. We have obtained a strong induction of EGFP only in male testes, which correlated with a high level of rtTA expression only in this organ. To overcome the problem of lack of rtTA protein in the transgenic rat brain, we have delivered this Tet system activator with lentiviral vectors into the dentate gyrus of hippocampus of transgenic EGFP rats. As a result, after systemic application of doxycycline we have obtained inducible, stable and restricted to the desired brain region expression of EGFP. An advantage of this strategy is that the transgene is located in the same genetic milieu in every cell of the transgenic organism. This is crucial to obtain uniform expression of the regulated gene within the target brain structure. Combination of rat transgenesis and lentiviral vectors is a novel approach enabling precise spatiotemporal regulation of genes of interest strictly in the brain structure of choice or in other tissues. genesis 47:274–280, 2009. © 2009 Wiley‐Liss, Inc.     

Functional lentiviral vectors for xeroderma pigmentosum gene therapy

Xeroderma pigmentosum (XP) is a genetic disease characterized by an autosomal-transmitted genodermatosis involving impaired DNA repair activity, where XP patients present severe sensitivity to sunlight (UVB radiation) and are highly victimized by skin cancer. Complementing XP genes by gene therapy is one potential strategy for helping XP patients. However, current viral-based protocols still lack long-term and stable expression, due to limited post-mitotic infection and gene silencing (in the case of retroviruses) or transient expression and activation of immune response (in the case of adenoviruses). Here we demonstrate that the use of third-generation lentiviral vectors can overcome some of these limitations, rescuing the aberrant phenotype in different categories of the disease (XPA, XPC and XPD). Our results show that lentiviruses are efficient tools to transduce XP fibroblasts and correct repair-defective cellular phenotypes by recovering proper gene expression, normal UV survival and unscheduled DNA synthesis after UV radiation. We propose lentiviral vectors as an attractive alternative for gene therapy protocols focusing on DNA repair genetic diseases.     

Biohydrometallurgical processing of solids: a patent review

Autotrophic as well as heterotrophic bacteria and fungi play an important role for the industrial recovery of metals from low-grade ore or, in general, from low-grade mineral resources. The same inorganic bacterial pathways that are responsible for huge and expensive corrosion problems can be used for economical biohydrometallurgical applications. Metals and metalloids can be microbially transformed by oxidation, reduction, alkylation, dealkylation, solubilization, and␣precipitation mechanisms. Biohydrometallurgy, a branch of classical metallurgy, is not as widely publicized as other areas of metallurgy, e.g. pyrometallurgical or hydrometallurgical processes. Since 1990, approximately 15 international patent applications concerning biohydrometallurgical techniques have been claimed under the Patent Cooperation Treaty in contrast to a large number of patents concerning pyro- and hydrometallurgical techniques. Nevertheless, it is a very important field of investigation, especially for the future when aspects of a sustainable development have to be considered. New processes to support this development are applicable, at least on a laboratory scale. Bacterial leaching processes for the recovery of metals from solid residues are applied for low-grade ore, and, more recently, for fly ash, galvanic sludges, or, in general, for industrial wastes. It is possible to recover leached metals and to recycle them in metal-manufacturing industries. In addition, by removing the metals from residues, the environmental quality is improved, and the material can be re-used for construction purposes. Received: 21 April 1997 / Received revision: 14 July 1997 / Accepted: 27 July 1997     

Phlebotomine vectors of the leishmaniases: a review

ABSTRACT. An account is given of work published during the past 10 years incriminating species of phlebotomine sandflies as vectors of Leishmania species which infect man.
An assessment is made of the degrees of certainty of the vectorial roles of eighty-one species and subspecies of sandflies (thirty-seven Old World and forty-four New World) in the transmission of twenty-nine leishmanial parasites of mammals. At least one species of sandfly is considered to be a proven vector of each of ten parasites.
Of the eighty-one sandfly taxa, evidence is judged to be sufficient to incriminate nineteen as proven vectors (eleven Phlebotomus species and eight Lutzomyia species or subspecies) and evidence for a further fourteen (nine Phlebotomus species and five Lutzomyia species or subspecies) is considered to be strong.
The suggested criteria for incrimination of a vector are anthropophily and common infection with the same leishmanial parasite as that found in man in the same place. More weight should be given to natural infections persisting after the digestion of a bloodmeal than those in the presence of blood. Supporting evidence is a concordance in the distribution of the fly and the disease in man, proof that the fly feeds regularly on the reservoir host, a flourishing development of the parasite in infected flies and the experimental transmission of the parasite by the bite of the fly.     

Neural development in human embryonic stem cells-applications of lentiviral vectors

The derivation of neural lineages from human embryonic stem cells (hESCs) in vitro is based largely on exposure of hESCs to exogenous signals and substrates, designed to mimic conditions in the developing embryo. However, selection of specific lineages and the discovery of gene function in human neural development may be enhanced by the ability to intrinsically regulate gene expression. Recombinant lentiviral vectors provide an efficient method to stably introduce genes into hESC and their differentiating derivatives. Here we review the methods used to derive neural cells from hESCs, transduction of these cells with lentiviral vectors, and improvements that have been made to the vectors to enhance viral integration and transgene expression. Finally, we explore prospects for future uses of lentiviral vectors in hESC research, including their applications in library screening for drug development, zinc finger nucleases for gene editing and optogenetics to interrogate cellular pathways and function.