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The majority of clinical trials for gene therapy currently employ retroviral-mediated gene delivery. This is because the life
cycle of the retrovirus is well understood and can be effectively manipulated to generate vectors that can be efficiently
and safely packaged. Here, we review the molecular technology behind the generation of recombinant retroviral vectors. We
also highlight the problems associated with the use of these viruses as gene therapy vehicles and discuss future developments
that will be necessary to maintain retroviral vectors at the forefront of gene transfer technology. 相似文献
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Retroviral vector particles displaying the antigen-binding site of an antibody enable cell-type-specific gene transfer. 总被引:12,自引:9,他引:3
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Retroviral vectors are the most efficient tool for stably introducing genes into vertebrate cells. However, their use is limited by the host range of the retrovirus from which they are derived. To alter the host range, we recently constructed retrovirus vector particles, derived from spleen necrosis virus, that display a single-chain antigen-binding site of an antibody (scA) on the viral surface (T.-H. T. Chu, I. Martinez, W. Sheay, and R. Dornburg, Gene Ther. 1:292-299, 1994). Using a hapten (2,4-dinitrophenol) model system, we showed that such particles are competent for infection. In this study, we repeated our experiments using an scA directed against a cell surface protein expressed on various human carcinoma cell lines. We found that such scA-displaying particles can efficiently infect human cells that express the corresponding antigen. Particles with wild-type spleen necrosis virus envelope are minimally infectious on such cells. The addition of the original monoclonal antibody to the viral vector particle solution prior to infection inhibited infection. This competition assay showed that the infection is mediated by the antibody moiety and, therefore, is antibody specific. These data indicate that retroviral vectors with antibody-envelope fusion proteins may be a valuable tool for selectively introducing genes into any target cell. 相似文献
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In-Seok Oh Ji-Chul Lee Myung-shin Lee Jun-ho Chung Dong-Myung Kim 《Bioprocess and biosystems engineering》2010,33(1):127-132
Botulinum neurotoxin serotype B (BoNT/B)-specific Fab was expressed in a cell-free protein synthesis system derived from an
E. coli extract. The cell-free synthesized antibody fragment was found to be effective in neutralizing the toxicity of BoNT/B in
animal studies. Expression of functional Fab required an appropriately controlled and stably maintained redox potential. Under
an optimized redox condition, the cell extract, whose disulfide reducing activity had been exhausted, could generate bio-functional
Fab molecules. Use of a cell extract enriched with molecular chaperones (GroEL/ES) and disulfide bond isomerases were effective
in obtaining larger quantities of functional Fab. Under the optimized reaction conditions, approximately 30 μg of functional
Fab was obtained after purification from 1 mL reaction mixture. 相似文献
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Retroviral vectors provide a safe and efficient method of introducing genes of therapeutic interest into dividing cells. The principle limitation of these vectors in the past has been poor gene expression in vivo. This problem has been overcome recently through the use of tissue-specific enhancers in commonly used retroviral vectors. In this review we discuss both the relevant biology and some of the practical applications of retroviral vectors in gene therapy. 相似文献
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Retroviral vectors for human gene delivery 总被引:7,自引:0,他引:7
The potential for gene therapy to cure a wide range of diseases has lead to high expectations and a great increase in research efforts in this area. At present, viral vectors are the most efficient means of delivering a corrective gene into human cells. While a number of different viral vectors are under development, retroviral vectors are currently the most common type used in clinical trials today. However, the production of retroviral vectors for gene therapy applications faces a number of challenges. Of primary concern is the low titre of vector stocks produced by packaging cells in culture and the inherent instability of retroviral vector activity. The problems facing large-scale retroviral vector production are outlined in this review and the research efforts by a number of groups who have attempted to optimise production methods are presented. 相似文献
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Efficient bacterial production of functional antibody fragments using a phagemid vector 总被引:2,自引:1,他引:1
The so-called ‘in vitro evolutionary method’ using a phage display system has been applied for protein engineering of the antigen-binding fragment of antibodies (Fab) by conducting random mutagenesis at the antigen-binding site in combination with antigen-based biopanning. However, isolated phage clones displaying Fab cannot necessarily be used for efficient bacterial production of engineered Fab proteins, often due to deleterious defects in their proper folding abilities derived in compensation for the gain of high affinity for a particular antigen. We here report a new method of an efficient and direct bacterial expression system for the phagemid-coded Fab proteins without use of the helper phage. To overcome a low folding efficiency derived from somatic hypermutations, if any, we have established optimum conditions for bacterial cultivation and protein expression, utilizing unusually long cultivation time (>50 h) and very low temperature (25 °C) and thereby leading to the production and extracellular secretion of Fab proteins in a very high yield (3–15 mg/L of culture). The purified Fab folded correctly and could efficiently bind an antigen, as judged by circular dichroism and isothermal titration calorimetry, respectively. 相似文献
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Toward highly efficient cell-type-specific gene transfer with retroviral vectors displaying single-chain antibodies. 总被引:4,自引:2,他引:2
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Recently, we constructed retroviral vector particles derived from spleen necrosis virus (SNV) that display a single-chain antibody (scA) on the viral surface. By transient transfection protocols, we showed that such particles are competent for infection and cell type specific. Efficient infection was dependent on the presence of wild-type envelope, although wild-type SNV was not infectious on target cells (T.-H. T. Chu and R. Dornburg, J. Virol. 69:2659-2663, 1995; T.-H. T. Chu, I. Martinez, W. C. Sheay, and R. Dornburg, Gene Ther. 1:292-299, 1994). In this study, stable packaging lines were constructed and detailed biological and biochemical studies were performed. Chimeric scA-envelope fusion proteins were expressed as efficiently as wild-type envelope and were stable over a period of at least 6 h. Only a fully functional wild-type envelope could act as a helper for efficient virus penetration. The ratio of wild-type envelope protein to chimeric envelope protein appears to determine the efficiency of infection. Virus titers of targeting vectors obtained from stable packaging lines were as high as 10(4) CFU/ml. A 25-fold concentration of vector virus stocks resulted in a 200-fold increase in virus titers (up to 10(6) CFU/ml). These data indicate that an inhibitor of infection was (at least partially) removed by the concentration protocol. Our data show that this technology has several variables for further improvements and, therefore, has the potential to become a powerful tool for cell-type-specific in vivo human gene therapy. 相似文献
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A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase. The applicability of the system is demonstrated by the synthesis of anti-lysozyme and anti-CD4 Fab antibody fragments yielding approximately 10 μg Fab per milliliter reaction mixture. The lack of endotoxins in this system is a prerequisite that synthesized Fab can be applied directly using whole synthesis reactions in cell-based assays that are sensitive to this substance class. Moreover, the system is compatible with PCR-generated linear templates enabling automated generation of antibody fragments in a high-throughput manner, and facilitating its application for screening and validation purposes. 相似文献
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Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus S protein 总被引:10,自引:0,他引:10
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Giroglou T Cinatl J Rabenau H Drosten C Schwalbe H Doerr HW von Laer D 《Journal of virology》2004,78(17):9007-9015
The worldwide outbreak of severe acute respiratory syndrome (SARS) was shown to be associated with a novel coronavirus (CoV) now called SARS CoV. We report here the generation of SARS CoV S protein-pseudotyped murine leukemia virus (MLV) vector particles. The wild-type S protein pseudotyped MLV vectors, although at a low efficiency. Partial deletion of the cytoplasmic tail of S dramatically increased infectivity of pseudotypes, with titers only two- to threefold lower than those of pseudotypes generated in parallel with the vesicular stomatitis virus G protein. S-pseudotyped MLV particles were used to analyze viral tropism. MLV(SARS) pseudotypes and wild-type SARS CoV displayed similar cell types and tissue and host restrictions, indicating that the expression of a functional receptor is the major restraint in permissiveness to SARS CoV infection. Efficient gene transfer could be detected in Vero and CaCo2 cells, whereas the level of gene marking of 293T, HeLa, and HepG2 cells was only slightly above background levels. A cat cell line and a dog cell line were not susceptible. Interestingly, PK-15, a porcine kidney cell line, and primary porcine kidney cells were also highly permissive for SARS S pseudotypes and wild-type SARS CoV. This finding suggests that swine may be susceptible to SARS infection and may be a source for infection of humans. Taken together, these results indicate that MLV(SARS) pseudotypes are highly valuable for functional studies of viral tropism and entry and, in addition, can be a powerful tool for the development of therapeutic entry inhibitors without posing a biohazard to human beings. 相似文献
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Antibody fragments can be isolated rapidly using techniques such as phage display and can be expressed to high levels in microbial systems. However, to date such antibody fragments have been of limited use for many therapeutic applications because they are rapidly cleared from the body. We present a strategy for the site-specific chemical modification of antibody fragments with polyethylene glycol, which results in the production of antibody fragments with long in vivo half-lives and full retention of antigen-binding properties. This technology should allow more rapid and economical production of therapeutic antibodies for chronic disease therapy. 相似文献
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Basu B Correa de Sampaio P Mohammed H Fogarasi M Corrie P Watkins NA Smethurst PA English WR Ouwehand WH Murphy G 《The international journal of biochemistry & cell biology》2012,44(2):393-403
The membrane associated MMP, MT1-MMP, is a critical pericellular protease involved in tumour cell invasion and angiogenesis and is highly up-regulated in numerous human cancers. It therefore represents an exciting new therapeutic cancer-specific target. We have generated recombinant human scFv antibodies against the non-catalytic, hemopexin domain of MT1-MMP that modulate its interactions with collagen. One of these is an effective inhibitor of the invasive capacity of cancer cells and of angiogenesis in model systems. This demonstrates that targeting sites outside the catalytic domain presents a potential novel approach to proteinase inhibition that could have applications in cancer therapeutics. 相似文献
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We have designed a new pathway for the synthesis of targeted polymeric drug delivery systems, using polymerizable antibody Fab' fragments (MA-Fab'). The targeted systems can be directly prepared by copolymerization of the MA-Fab', N-(2-hydroxypropyl)methacrylamide (HPMA) and drug-containing monomers. Both MA-Fab' and the Fab'-targeted copolymers can effectively bind to target cells. An MA-Fab' (from OV-TL 16 Ab) targeted HPMA copolymer containing mesochlorin e6 (Mce6) was synthesized by copolymerization of MA-Fab', HPMA, and MA-GFLG-Mce6. The targeted copolymer exhibited a higher cytotoxicity toward OVCAR-3 human ovarian carcinoma cells than the nontargeted Mce6-containing copolymer or free Mce6. The targeted copolymer was internalized more efficiently by OVCAR-3 cells than the nontargeted copolymer. 相似文献
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M Ayala M Due?as A Santos J Vázquez A Menéndez A Silva J V Gavilondo 《BioTechniques》1992,13(5):790-799
We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA. 相似文献
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