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Leukemia Jul 2018Viral vectors provide an efficient means for modification of eukaryotic cells, and their use is now commonplace in academic laboratories and industry for both research... (Review)
Review
Viral vectors provide an efficient means for modification of eukaryotic cells, and their use is now commonplace in academic laboratories and industry for both research and clinical gene therapy applications. Lentiviral vectors, derived from the human immunodeficiency virus, have been extensively investigated and optimized over the past two decades. Third-generation, self-inactivating lentiviral vectors have recently been used in multiple clinical trials to introduce genes into hematopoietic stem cells to correct primary immunodeficiencies and hemoglobinopathies. These vectors have also been used to introduce genes into mature T cells to generate immunity to cancer through the delivery of chimeric antigen receptors (CARs) or cloned T-cell receptors. CAR T-cell therapies engineered using lentiviral vectors have demonstrated noteworthy clinical success in patients with B-cell malignancies leading to regulatory approval of the first genetically engineered cellular therapy using lentiviral vectors. In this review, we discuss several aspects of lentiviral vectors that will be of interest to clinicians, including an overview of lentiviral vector development, the current uses of viral vectors as therapy for primary immunodeficiencies and cancers, large-scale manufacturing of lentiviral vectors, and long-term follow-up of patients treated with gene therapy products.
Topics: Animals; Clinical Studies as Topic; Gene Transfer Techniques; Genetic Engineering; Genetic Therapy; Genetic Vectors; Humans; Lentivirus; Transduction, Genetic
PubMed: 29654266
DOI: 10.1038/s41375-018-0106-0 -
Viruses Feb 2021Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and... (Review)
Review
Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.
Topics: Animals; Bioreactors; Cell Culture Techniques; Cell Line; Genetic Vectors; Humans; Lentivirus; Transduction, Genetic; Viral Envelope Proteins; Virus Cultivation
PubMed: 33572347
DOI: 10.3390/v13020268 -
Journal of Virology Aug 1996We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helper-free retrovirus with titers of 1 X 10(6) to 5 X 10(6)...
We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helper-free retrovirus with titers of 1 X 10(6) to 5 X 10(6) within 48 h. These vectors contain the immediate early region of the human cytomegalovirus enhancer-promoter fused to the Moloney murine leukemia virus long terminal repeat at the TATA box in the 5' U3 region, yielding the pCL promoter. By selecting vectors designed to express genes from one of four promoters (dihydrofolate reductase, Rous sarcoma virus, long terminal repeat, or cytomegalovirus), the pCL system permits the investigator to control the level of gene expression in target cells over a 100-fold range, while maintaining uniformly high titers of virus from transiently transfected producer cells. The pCL packaging plasmids lack a packaging signal (delta-psi) and include an added safety modification that renders them self-inactivating through the deletion of the 3' U3 enhancer. Ecotropic, amphotropic (4070A), and amphotropic-mink cell focus-forming hybrid (10A1) envelope constructions have been prepared and tested, permitting flexible selection of vector pseudotype in accordance with experimental needs. Vector supernatants are free of helper virus and are of sufficiently high titer within 2 days of transient transfection in 293 cells to permit infection of more than 50% of randomly cycling target cells in culture. We demonstrated the efficacy of these vectors by using them to transfer three potent cell cycle control genes (the p16(INK4A), p53, and Rb1 genes) into human glioblastoma cells.
Topics: DNA, Recombinant; DNA, Viral; Genetic Vectors; Humans; Retroviridae
PubMed: 8764092
DOI: 10.1128/JVI.70.8.5701-5705.1996 -
Viruses Jul 2022Lentiviral vectors (LV) have been developed upon knowledge accumulated in the virology field, in particular intensive research on HIV biology since its discovery in 1983...
Lentiviral vectors (LV) have been developed upon knowledge accumulated in the virology field, in particular intensive research on HIV biology since its discovery in 1983 [...].
Topics: Genetic Vectors; Lentivirus
PubMed: 35891475
DOI: 10.3390/v14071492 -
Current Opinion in Virology Dec 2016Hematopoietic stem cell (HSC) gene therapy using retroviral vectors is a powerful and promising approach to permanently correct many hematopoietic disorders. Increasing... (Review)
Review
Hematopoietic stem cell (HSC) gene therapy using retroviral vectors is a powerful and promising approach to permanently correct many hematopoietic disorders. Increasing the transduction of quiescent HSCs and reducing genotoxicity are major challenges in the field. Retroviral vectors, including lentiviral and foamy vectors, have been extensively modified resulting in improved safety and efficacy. This review will focus on recent advances to improve vector entry, transduction efficiency, control of transgene expression and approaches to improve safety by modifying the retroviral integration profile.
Topics: Animals; DNA Damage; Genetic Therapy; Genetic Vectors; Hematologic Diseases; Hematopoietic Stem Cells; Humans; Retroviridae; Transduction, Genetic
PubMed: 27521874
DOI: 10.1016/j.coviro.2016.07.010 -
Drug News & Perspectives Nov 2008The co-expression of genes can greatly enhance the efficiency and versatility of gene therapy applications. A variety of options exists to simultaneously express two... (Review)
Review
The co-expression of genes can greatly enhance the efficiency and versatility of gene therapy applications. A variety of options exists to simultaneously express two genes in genetically modified cells. The most common approach relies on bicistronic vectors in which the genes are linked to each other by an internal ribosome entry site allowing co-translational expression of both cistrons. This review gives an overview of gene vector systems currently in use for coordinated expression of transgenes with particular focus on retroviral bicistronic vectors. The structural and functional characteristics of bicistronic vectors are discussed as well as factors influencing their efficiency such as the nature of the transgenes and the cell types in which they are expressed. Finally, the potential for clinical applications of retroviral bicistronic vectors in molecular genetics is highlighted.
Topics: Animals; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Retroviridae; Transduction, Genetic; Transfection; Transgenes
PubMed: 19180265
DOI: 10.1358/dnp.2008.21.9.1290817 -
Human Gene Therapy Mar 2016Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used,...
Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34(+) cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy.
Topics: Cell Line; Enhancer Elements, Genetic; Gene Order; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Hematopoietic Stem Cells; Humans; Insulator Elements; Plasmids; Spumavirus; Transduction, Genetic; Virus Integration; Virus Replication
PubMed: 26715244
DOI: 10.1089/hum.2015.110 -
Gene Therapy Feb 2021Lentiviral vectors have become popular tools for stable genetic modification of mammalian cells. In some applications of lentiviral vector-transduced cells,...
Lentiviral vectors have become popular tools for stable genetic modification of mammalian cells. In some applications of lentiviral vector-transduced cells, infectious-lentiviral particles should be absent. Quantification of the free-vector particles that remain from the inoculum can be difficult. Therefore a formula was established that yields an estimation of the 'Reduction Ratio.' This ratio represents the loss of titer based on a number of vector-inactivating effects. In this study, we evaluated several parameters and assumptions that were used in the current formula. We generated new data on the stability and trypsin sensitivity of lentiviral vectors pseudotyped with eight heterologous envelope proteins and the loss of vectors by washing or passaging the cell cultures. Our data demonstrate that the loss of virus titer under the influence of trypsin as well as the half-life of the particles in tissue culture medium is dependent on the vector's envelope protein. While VSV-G-envelope-pseudotyped particles were unsensitive to trypsin, the titer of vectors pseudotyped with other envelope proteins decreased 2-110-fold. The half-life in culture medium ranged from 8 to 40 h for the different envelope-pseudotyped vectors, with 35 h for VSV-G-envelope-pseudotyped vector particles. Additionally, we found that removal of the culture medium from Ø35 mm to Ø10 cm dishes reduces the amount of vector particles in the culture by 50-fold and 20-fold, respectively. Together these data can be used to more precisely estimate the maximum number of free lentiviral vector particles in cell cultures.
Topics: Animals; Genetic Vectors; Lentivirus; Transduction, Genetic; Viral Envelope Proteins
PubMed: 32973351
DOI: 10.1038/s41434-020-00193-y -
Biology Open Feb 2022Genetic manipulation of primary lymphocytes is crucial for both clinical purposes and fundamental research. Despite their broad use, we encountered a paucity of data on...
Genetic manipulation of primary lymphocytes is crucial for both clinical purposes and fundamental research. Despite their broad use, we encountered a paucity of data on systematic comparison and optimization of retroviral vectors, the workhorses of genetic modification of primary lymphocytes. Here, we report the construction and validation of a versatile range of retroviral expression vectors. These vectors can be used for the knockdown or overexpression of genes of interest in primary human and murine lymphocytes, in combination with a wide choice of selection and reporter strategies. By streamlining the vector backbone and insert design, these publicly available vectors allow easy interchangeability of the independent building blocks, such as different promoters, fluorescent proteins, surface markers and antibiotic resistance cassettes. We validated these vectors and tested the optimal promoters for in vitro and in vivo overexpression and knockdown of the murine T cell antigen receptor. By publicly sharing these vectors and the data on their optimization, we aim to facilitate genetic modification of primary lymphocytes for researchers entering this field.
Topics: Animals; Genetic Vectors; Humans; Lymphocytes; Mice; Promoter Regions, Genetic; Retroviridae
PubMed: 35229875
DOI: 10.1242/bio.059032 -
Gene Nov 2021Although seen as a revolution in modern science, gene therapy has been plagued by failed clinical trials and controversial ethics in the last thirty years. Moreover,... (Review)
Review
Although seen as a revolution in modern science, gene therapy has been plagued by failed clinical trials and controversial ethics in the last thirty years. Moreover, there is no comprehensive, in-depth, high-quality analysis of global gene therapy patents. This paper proposes a method to correctly retrieve patents to address the issue and use it for the patent landscape. The results show the global patent landscape of gene therapy, with the United States dominating the field, while China has emerged as a leader in recent years. For various reasons, the EU, Korea, and Japan lag in the development of patented technologies. China has edged closer to the US in both live and indefinite patents, with the Chinese Academy of Military Medical Sciences and the Chinese Academy of Sciences leading the way, surpassing primary applicants such as the US Department of Health and Human Services, the University of California, and the University of Pennsylvania. The study also reveals four broad categories of technologies that have been extensively studied in gene therapy: basic biology of the gene and diseases, diseases being treated, gene delivery methods, and potential adverse events. What is more, Adeno-Associated Virus, Retrovirus, and Lentivirus are the most prevalent gene therapy delivery vectors after 2014. The industrial development trend revealed in this paper can provide an evidence-based basis for scientific research management and decision-making.
Topics: China; Dependovirus; European Union; Genetic Therapy; Genetic Vectors; Humans; Japan; Lentivirus; Patents as Topic; Republic of Korea; Retroviridae; United States
PubMed: 34371094
DOI: 10.1016/j.gene.2021.145889