共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
Production and purification of lentiviral vectors 总被引:1,自引:0,他引:1
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. 相似文献
3.
Transgenic HIV-1-derived lentiviral particles are at the forefront of current gene therapy and tissue engineering initiatives, which will require optimal protocols for large-scale production of clinical-grade therapeutic lentiviruses. Production of latest-generation self-inactivating lentiviral particles requires cotransfection of mammalian production cell lines with two helper plasmids along with the lentivector, whose transgene-encoding expression cassette is the only genetic information stably transduced into target chromosomes. Capitalizing on a recently designed lentiviral expression vector family, we conducted rigorous analysis of production-relevant parameters including transfection, cell density, media composition, temperature, relative (helper) vector concentrations and genetic configuration. Comparative analysis of lentiviral particle performance (VP) was based on the viral titer (reflecting the number of transduction-competent lentiviral particles) relative to the number of lentiviral particles produced (correlating with p24 production levels) (VP=titer/viral particle number). Optimal lentiviral production parameters, resulting in up to 132-fold greater VP compared to standard protocols, required (i) CaPO4-based transfection (ii) of helper plasmids and lentivector at a fixed concentration ratio (helper plasmid I:helper plasmid II:lentivector=1:1:2) (iii) into 1x10(5) human embryonic kidney cells/cm2 (HEK293-T) (iv) cultivated at 37 degrees C (v) in Advanced D-MEM medium supplemented with (vi) 2% fetal calf serum, (vii) and a culture additive containing 0.01 mM cholesterol, 0.01 mM egg's lecithin and 1x chemically defined lipid concentrate. (viii) Furthermore, constitutive transgene expression units placed in a forward polyadenylation site (pA)-free orientation relative to the lentivector backbone resulted in optimal transgene transduction/expression. Our studies suggest that detailed knowledge of lentivector design and the production of lentiviral particles will advance large-scale manufacturing of clinically relevant lentiviruses for future gene therapy applications. 相似文献
4.
5.
Adenoviral gene transfer of eNOS: high-level expression in ex vivo expanded marrow stromal cells 总被引:3,自引:0,他引:3
Deng W Bivalacqua TJ Chattergoon NN Hyman AL Jeter JR Kadowitz PJ 《American journal of physiology. Cell physiology》2003,285(5):C1322-C1329
Endothelial nitric oxide synthase (eNOS) is an attractive target for cardiovascular gene therapy. Marrow stromal cells (MSCs), also known as mesenchymal stem cells, hold great promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated eNOS gene transfer into ex vivo expanded MSCs, rat MSCs (rMSCs) were isolated, expanded ex vivo, and transduced with Ad5RSVeNOS, an adenoviral vector containing the eNOS gene under the control of the Rous sarcoma virus promoter. The presence of eNOS protein in Ad5RSVeNOS-transduced rMSCs was confirmed by immunohistochemical and Western blot analysis. Transduction efficiency was dose dependent, and eNOS transgene expression in rMSCs persisted for =" BORDER="0">21 days in culture. The rMSCs retained multipotential differentiation capability after adenoviral-mediated eNOS gene transfer. Furthermore, intracavernosal injection of Ad5RSVeNOS-transduced rMSCs increased the expression of eNOS in the corpus cavernosum, and stem cells were identified within corporal sinusoids. These findings demonstrate that replication-deficient recombinant adenovirus can be used to engineer ex vivo expanded rMSCs and that high-level eNOS transgene expression can be achieved, pointing out the clinical potential of using this novel adult stem cell-based gene therapy method for the treatment of cardiovascular diseases. adenoviral vector; nitric oxide; gene expression; differentiation; gene therapy 相似文献
6.
Dynamics of transgene expression in a neural stem cell line transduced with lentiviral vectors incorporating the cHS4 insulator 总被引:2,自引:0,他引:2
Jakobsson J Rosenqvist N Thompson L Barraud P Lundberg C 《Experimental cell research》2004,298(2):611-623
Transplantation of genetically manipulated cells to the central nervous system holds great promise for the treatment of several severe neurological disorders. The success of this strategy relies on sufficient levels of transgene expression after transplantation. This has been difficult to achieve, however, due to transgene silencing. In this study, we transduced the neural stem cell line RN33B with self-inactivating lentiviral vectors and analyzed transgenic expression of green fluorescent protein (GFP) in several different settings both in vitro and after transplantation to the brain. We found that the transgene was affected of silencing both when transduced cells were proliferating and after differentiation. To prevent silencing, the cHS4 insulator was incorporated into the lentiviral vector. We found that a vector carrying the cHS4 insulator was partially protected against differentiation-dependent downregulation in vitro and in vivo. However, in proliferating cells, we found evidence for variegation and positional effects that were not prevented by the cHS4 insulator, suggesting that the mechanism behind silencing in proliferating cells is not the same mechanism influencing differentiation-dependent silencing. Taken together, these findings favor vector optimization as a strategy for achieving efficient ex vivo gene transfer in the central nervous system. 相似文献
7.
Prolonged adherence of human immunodeficiency virus-derived vector particles to hematopoietic target cells leads to secondary transduction in vitro and in vivo
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Human immunodeficiency virus type 1-derived lentivirus vectors bearing the vesicular stomatitis virus G (VSV-G) envelope glycoprotein demonstrate a wide host range and can stably transduce quiescent hematopoietic stem cells. In light of concerns about biosafety and potential germ line transmission, they have been used predominantly for ex vivo strategies, thought to ensure the removal of excess surface-bound particles and prevent in vivo dissemination. Studies presented here instead reveal prolonged particle adherence after ex vivo exposure, despite serial wash procedures, with subsequent transduction of secondary target cells in direct and transwell cocultures. We explored the critical parameters affecting particle retention and transfer and show that attachment to the cell surface selectively protects virus particles from serum complement-mediated inactivation. Moreover, studies with nonmyeloablated murine recipients show that transplantation of vector-exposed, washed hematopoietic cells results in systemic dissemination of functional VSV-G/lentivector particles. We demonstrate genetic marking by inadvertent transfer of vector particles and prolonged expression of transgene product in recipient tissues. Our findings have implications for biosafety, vector design, and cell biology research. 相似文献
8.
In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors 总被引:9,自引:0,他引:9
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Ulrich Kuhn Atsushi Terunuma Wolfgang Pfutzner Ruth Ann Foster Jonathan C. Vogel 《Journal of virology》2002,76(3):1496-1504
For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture. 相似文献
9.
10.
In order to optimize viral gene transfer into hematopoietic stem cells we developed retroviral and lentiviral vectors with B cell-specificity. Using fragments of the human CD19 promoter we demonstrate in mice that upon lethal irradiation and reconstitution with virus-treated bone marrow transgene expression is specific for the B cell-lineage. We compare various viral constructs with different promoter length and with or without B cell-specific enhancer regions in retro- and lentiviral backbones. Our data suggest that B cell-targeting for gene therapy approaches is feasible, leads to stable expression, and can be modulated by using different transduction and expression systems. 相似文献
11.
Alpha-1 antitrypsin (AAT) deficiency is a lethal hereditary disorder characterized by a severe diminution in plasma levels of AAT leading to progressive liver dysfunction. Since mesenchymal stem cells can differentiate into hepatocyte-like cells they offer a potential unlimited source in autologous transplant procedures. The transfer of genetically modified hepatocyte cells derived from hMSCs into the body constitutes a novel paradigm of coupling cell therapy with gene therapy for this disease. hMSCs were isolated by density gradient centrifugation and plastic adherence. Hepatic differentiation was induced by exposing hMSC to induction medium for up to 21 days. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR and immunocytochemistry. The chimeric AAT-Jred transgene was transferred to differentiated cells using a lentiviral vector and its expression was visualized by fluorescent microscopy. Flow cytometric analysis confirmed that hMSCs were obtained. Major hepatocyte marker genes expression were confirmed by RT-PCR and immunocytochemistry. AAT gene was successfully introduced into hepatocyte-like cells differentiated from hMSCs. This established system could be suitable for generation of hMSC derived hepatocyte-like cells containing the normal AAT gene, thus offering a potential in vitro source of cells for transplantation therapy of liver diseases in AAT-deficient patients. 相似文献
12.
Bemelmans AP Bonnel S Houhou L Dufour N Nandrot E Helmlinger D Sarkis C Abitbol M Mallet J 《The journal of gene medicine》2005,7(10):1367-1374
13.
含GDNF基因的慢病毒载体的构建和其在人胚胎神经干细胞中的表达 总被引:1,自引:0,他引:1
利用含胶质源性神经营养因子(Glial cell derived neurotrophic factor, GDNF)基因的慢病毒(Lentivirus)载体转染了人胚胎来源的神经干细胞, 探讨了转染后GDNF在神经干细胞中的体外表达水平及其影响因素。首先GDNF基因被克隆入慢病毒载体, 通过瞬时转染法包装出病毒上清, 经滴度鉴定后分别按拷贝数分别为 1、2.5、5、10转染神经干细胞。转染后细胞经过潮霉素筛选得到均一表达GDNF的神经干细胞体系。其后分别利用酶联免疫吸附(ELISA)方法和Real-time PCR方法测定不同转染组细胞在不同时间点GDNF的蛋白分泌水平和基因表达水平。实验中构建了表达GDNF基因的慢病毒载体, 包装出的病毒上清在体外培养条件下成功转染了神经干细胞, 经潮霉素筛选可以得到均一的持续表达分泌GDNF的人胚胎皮层神经干细胞体系。实验结果表明转染拷贝数可以影响GDNF的分泌水平, 相同条件下转染拷贝数越高, GDNF分泌量越多, 其基因表达水平越高。因此, 含GDNF的慢病毒载体可以成功转染人胚胎来源的神经干细胞, 使其持续表达GDNF, 转染过程中可以通过拷贝数在一定水平上控制GDNF的蛋白分泌水平和基因表达水平。 相似文献
14.
Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4-7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments. 相似文献
15.
16.
17.
Adeno-associated virus type 2-mediated transduction of human monocyte-derived dendritic cells: implications for ex vivo immunotherapy 总被引:6,自引:0,他引:6
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Dendritic cells (DCs) are pivotal antigen-presenting cells for regulating immune responses. A major focus of contemporary vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that ameliorate disease states as diverse as malignancy, infection, autoimmunity, and allergy. The present study has evaluated adeno-associated virus (AAV) type 2 as a vector for ex vivo gene transfer to human peripheral blood monocyte (MO)-derived DCs. AAV is a nonpathogenic parvovirus that infects a wide variety of human cell lineages in vivo and in vitro, for long-term transgene expression without requirements for cell proliferation. The presented data demonstrate that recombinant AAV (rAAV) can efficiently transduce MOs as well as DCs generated by MO culture with granulocyte-macrophage colony-stimulating factor plus interleukin in vitro. rAAV transgene expression in MO-derived DCs could be enhanced by etoposide, previously reported to enhance AAV gene expression. rAAV transduction of freshly purified MO followed by 7 days of culture with cytokines to generate DCs, and subsequent sorting for coexpression of DC markers CD1a and CD40, showed robust transgene expression as well as evidence of nuclear localization of the rAAV genome in the DC population. Phenotypic analyses using multiple markers and functional assays of one-way allogeneic mixed leukocyte reactions indicated that rAAV-transduced MO-derived DCs were as equivalent to nontransduced DCs. These results support the utility of rAAV vectors for future human DC vaccine studies. 相似文献
18.
Gagnoux-Palacios L Hervouet C Spirito F Roques S Mezzina M Danos O Meneguzzi G 《The journal of gene medicine》2005,7(9):1178-1186
BACKGROUND: Genetically modified keratinocytes generate transplantable self-renewing epithelia suitable for delivery of therapeutic polypeptides. However, the variety of viral vectors and experimental conditions currently used make fragmented or contradictory the information on the transduction efficiency of the human primary keratinocytes. To compare the suitability of the most currently used viral vectors for efficient gene transfer to human keratinocytes, we have performed a comparative study using a panel of recombinant constructs. METHODS: For each vector, the transduction efficiency and the persistence of the transgene expression were quantified by fluorescence microscopy and flow cytometry analysis of the infected cells. RESULTS: We show that: (1) canine and human adenoviral vectors achieve a highly efficient but transient transduction of both primary and immortalized keratinocytes; (2) the adenovirus-associated virus (AAV) vectors transduce immortalized keratinocytes, albeit with a short-lived gene expression (<4 days), but fail to infect primary keratinocytes; and (3) under appropriate conditions, the oncoretroviral and lentiviral vectors can permanently transduce up to 100% of primary keratinocytes, but the highly clonogenic keratinocytes are more efficiently targeted by lentiviral vectors. CONCLUSIONS: Therefore, AAV vectors are unsuitable to transduce primary keratinocytes, while human and canine adenoviral vectors appears to be appropriate to achieve short-term delivery of therapeutic products. Recombinant retroviruses provide sustained expression of the transgene, but the lentiviral vectors are the most suitable for ex vivo gene therapy because of their ability to transduce clonogenic primary keratinocytes. 相似文献
19.
Kyriakou CA Yong KL Benjamin R Pizzey A Dogan A Singh N Davidoff AM Nathwani AC 《The journal of gene medicine》2006,8(3):253-264
BACKGROUND: Efficient gene transfer to bone marrow derived mesenchymal stem cells (MSC) would provide an important opportunity to express potent anticancer agents in the tumour microenvironment because of their contribution to the tumour stroma. METHODS: HIV-based lentiviral vectors were pseudotyped with four different envelope proteins; amphotropic murine leukaemia virus (ampho), murine leukaemia virus (10A1), feline endogenous virus (RD114), and the vesicular stomatitis virus glycoprotein (VSVG). These pseudotypes were examined for transduction efficiency in human bone marrow derived MSC. The effect of lentiviral expression of truncated soluble vascular endothelial growth factor decoy receptor (tsFlk-1) in MSC on growth of Raji cells was determined, both in vitro and in vivo. RESULTS: All lentiviral vectors produced significant levels of transduction at an multiplicity of infection (MOI) of 1, those bearing the RD114 envelope glycoprotein consistently produced higher transduction levels (mean 70 +/- 6%) compared with the other pseudotyped lentiviral vectors, although there was significant inter-donor variation. Stable transgene expression was achieved after multiple rounds of transduction with VSVG-pseudotyped particles, without alteration in the differentiative capacity of transduced cells. Co-injection of MSC stably expressing tsFlk-1 with Raji Burkitt's lymphoma cells significantly impaired subcutaneous tumour growth in immunodeficient mice when compared to controls where either unmanipulated MSC or GFP-expressing MSC were used. CONCLUSIONS: Human MSC are easily transduced by pseudotyped lentiviral particles but there is inter-donor variation in transduction efficiency. Gene-modified MSC expressing a gene of therapeutic potential can moderate growth of haematological malignancies. 相似文献