Journal articles on the topic 'Lentiviral vector'
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1
Yew, Chee-Hong Takahiro, Narmatha Gurumoorthy, Fazlina Nordin, Gee Jun Tye, Wan Safwani Wan Kamarul Zaman, Jun Jie Tan, and Min Hwei Ng. "Integrase deficient lentiviral vector: prospects for safe clinical applications." PeerJ 10 (August 12, 2022): e13704. http://dx.doi.org/10.7717/peerj.13704.
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HIV-1 derived lentiviral vector is an efficient transporter for delivering desired genetic materials into the targeted cells among many viral vectors. Genetic material transduced by lentiviral vector is integrated into the cell genome to introduce new functions, repair defective cell metabolism, and stimulate certain cell functions. Various measures have been administered in different generations of lentiviral vector systems to reduce the vector’s replicating capabilities. Despite numerous demonstrations of an excellent safety profile of integrative lentiviral vectors, the precautionary approach has prompted the development of integrase-deficient versions of these vectors. The generation of integrase-deficient lentiviral vectors by abrogating integrase activity in lentiviral vector systems reduces the rate of transgenes integration into host genomes. With this feature, the integrase-deficient lentiviral vector is advantageous for therapeutic implementation and widens its clinical applications. This short review delineates the biology of HIV-1-erived lentiviral vector, generation of integrase-deficient lentiviral vector, recent studies involving integrase-deficient lentiviral vectors, limitations, and prospects for neoteric clinical use.
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Li, Chen, Biao Qian, Zhao Ni, Qinzhang Wang, Zixiong Wang, Luping Ma, Zhili Liu, Qiang Li, and Xinmin Wang. "Construction of recombinant lentiviral vector containing human stem cell leukemia gene and its expression in interstitial cells of cajal." Open Life Sciences 15, no. 1 (March 25, 2020): 83–91. http://dx.doi.org/10.1515/biol-2020-0010.
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AbstractThis study aims to construct recombinant lentiviral vectors containing the human stem cell leukemia (SCL) gene and investigate their in vitro transfection efficiency in Interstitial Cells of Cajal (ICC) of guinea pig bladders. In this study, the human SCL gene was successfully cloned, and the recombinant lentivirus GV287-SCL was successfully constructed. The titer of the recombinant lentivirus was 5 × 108 TU /mL. After transfecting the ICCs with the lentiviral vector at different MOIs, the optimal MOI was determined to be 10.0, and the optimal transfection time was determined to be 3 days. The amplification product of the lentivirus transfection group was consistent with the target fragment, indicating that the SCL gene had been successfully introduced into ICCs. In conclusion, the recombinant lentiviral vector GV287-SCL was successfully constructed and transfected into the in vitro cultured ICCs. The successful expression of SCL in ICCs may provide an experimental basis for the in vivo transfection of the SCL gene.
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Perry, Christopher, and Andrea C. M. E. Rayat. "Lentiviral Vector Bioprocessing." Viruses 13, no. 2 (February 9, 2021): 268. http://dx.doi.org/10.3390/v13020268.
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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.
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Wang, Nan, Narendiran Rajasekaran, Tieying Hou, and Elizabeth Mellins. "Comparison of protein expression by different lentiviral vectors (51.12)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 51.12. http://dx.doi.org/10.4049/jimmunol.188.supp.51.12.
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Abstract HIV1-derived lentiviral vectors have been widely used as gene delivery tools due to their potent transduction capacity and stable expression after chromosomal integration in dividing and non-dividing mammalian cells. Lentiviral vectors were screened for expressing the murine class II chaperone, invariant chain (Ii), in hematopoietic stem and progenitor cells (HSPC). We compared various lentiviral vectors using GFP as a reporter gene in 293T cells and HSPC. We assessed a dual promoter vector (DP) with separate promoters for Ii and GFP, a fusion protein vector (FU) that expresses Ii and GFP together under a single promoter, and T2A vector (T2A) in which Ii and GFP are separated by a self-cleaving 2A peptide. Similar percentages of 293T cells were positive for Ii expression after being directly transfected with DP and T2A. The percentage was slightly higher in FU transfected cells. 293T cells transfected with DP have the highest GFP expression. We packaged each vector into lentivirus and found that both Ii and GFP expression levels dropped in 293T cells. Further reduction in expression was seen in viral transduced HSPC. The trend toward different expression levels of Ii and GFP persisted whether lentiviral transduction or vector transfection was used. Only DP and T2A constructs, but not FU constructs, resulted in functional Ii expression. Based on these results, we hypothesize that the poor expression of Ii may be due to deleterious effects of Ii on assembly of competent virus.
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Nguyen, Tuan Huy, Tatiana Khakhoulina, Andrew Simmons, Philippe Morel, and Didier Trono. "A Simple and Highly Effective Method for the Stable Transduction of Uncultured Porcine Hepatocytes Using Lentiviral Vector." Cell Transplantation 14, no. 7 (August 2005): 489–96. http://dx.doi.org/10.3727/000000005783982828.
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Gene therapy is an attractive approach for the treatment of a wide spectrum of liver diseases. Lentiviral vectors allow the stable integration of transgenes into the genome of nondividing differentiated cells including hepatocytes and could provide long-lasting expression of a therapeutic gene. To develop such approaches, preclinical studies in large animal models such as pigs are necessary to evaluate the feasibility and safety of stable lentiviral integration and long-term vector expression. In addition, effective lentivector-mediated gene transfer onto porcine hepatocytes could advance in cell-based therapies for acute liver failure. To investigate this issue, porcine hepatocytes were transduced in suspension immediately after their isolation in University of Wisconsin (UW) solution containing vitamin E. Up to 80% of hepatocytes stably expressed a GFP transgene after a single exposure to lentiviral vector coding for GFP under the control of either liver-specific or ubiquitous promoters. Moreover, porcine hepatocytes cryopreserved in UW solution containing fetal bovine serum, dimethyl sulfoxide, and vitamin E remained highly transducible with lentiviral vector after thawing. When thawed, transduced in suspension, and immediately transplanted into the spleen of immunodeficient mice, ex vivo lentivirally transgene marked xenogeneic hepatocytes were detected in murine liver. We demonstrated that porcine hepatocytes are highly susceptible to lentiviral vector and describe an easy methodology to efficiently, rapidly, and stably introduce transgenes into uncultured porcine hepatocytes.
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Kubo, Shuji, and Kohnosuke Mitani. "A New Hybrid System Capable of Efficient Lentiviral Vector Production and Stable Gene Transfer Mediated by a Single Helper-Dependent Adenoviral Vector." Journal of Virology 77, no. 5 (March 1, 2003): 2964–71. http://dx.doi.org/10.1128/jvi.77.5.2964-2971.2003.
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ABSTRACT To achieve efficient and sustained gene expression, we developed a new lentivirus/adenovirus hybrid vector (LA vector) that encodes sequences required for production of a human immunodeficiency virus-based lentiviral vector (i.e., a lentiviral vector, a gag/pol/rev expression cassette, a tetracycline-inducible envelope cassette, and the tetracycline-inducible transcriptional activator cassette) in a single helper-dependent adenovirus vector backbone. Via either transfection or infection, human cell lines transduced with the LA vector produced a lentiviral vector in a doxycycline-dependent manner at titers up to 105 to 106 green fluorescent protein transducing units per ml, which are comparable to the titers obtained by conventional multiple plasmid transfection methods. Efficient spread and persistent expression of the transgene were observed in cells maintained in long-term culture that had been infected with the LA vector. Furthermore, when cocultured with adherent cells infected with the LA vector, the human T-cell leukemia cell line was successfully transduced with a marker gene. This LA vector possesses the advantages of efficient gene transfer from an adenoviral vector and stable integration from a lentiviral vector; therefore, it might have potential for a variety of gene therapy applications.
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Sakuma, Toshie, Michael A. Barry, and Yasuhiro Ikeda. "Lentiviral vectors: basic to translational." Biochemical Journal 443, no. 3 (April 16, 2012): 603–18. http://dx.doi.org/10.1042/bj20120146.
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More than two decades have passed since genetically modified HIV was used for gene delivery. Through continuous improvements these early marker gene-carrying HIVs have evolved into safer and more effective lentiviral vectors. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production. Accordingly, lentivector technologies now have widespread use in basic biology and translational studies for stable transgene overexpression, persistent gene silencing, immunization, in vivo imaging, generating transgenic animals, induction of pluripotent cells, stem cell modification and lineage tracking, or site-directed gene editing. Moreover, in the present high-throughput ‘-omics’ era, the commercial availability of premade lentiviral vectors, which are engineered to express or silence genome-wide genes, accelerates the rapid expansion of this vector technology. In the present review, we assess the advances in lentiviral vector technology, including basic lentivirology, vector designs for improved efficiency and biosafety, protocols for vector production and infection, targeted gene delivery, advanced lentiviral applications and issues associated with the vector system.
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Breckpot, Karine, David Escors, Frederick Arce, Lucienne Lopes, Katarzyna Karwacz, Sandra Van Lint, Marleen Keyaerts, and Mary Collins. "HIV-1 Lentiviral Vector Immunogenicity Is Mediated by Toll-Like Receptor 3 (TLR3) and TLR7." Journal of Virology 84, no. 11 (March 17, 2010): 5627–36. http://dx.doi.org/10.1128/jvi.00014-10.
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ABSTRACT Lentiviral vectors are promising vaccine vector candidates that have been tested extensively in preclinical models of infectious disease and cancer immunotherapy. They are also used in gene therapy clinical trials both for the ex vivo modification of cells and for direct in vivo injection. It is therefore critical to understand the mechanism(s) by which such vectors might stimulate the immune system. We evaluated the effect of lentiviral vectors on myeloid dendritic cells (DC), the main target of lentiviral transduction following subcutaneous immunization. The activation of DC cultures was independent of the lentiviral pseudotype but dependent on cell entry and reverse transcription. In vivo-transduced DC also displayed a mature phenotype, produced tumor necrosis factor alpha (TNF-α), and stimulated naive CD8+ T cells. The lentiviral activation of DC was Toll-like receptor (TLR) dependent, as it was inhibited in TRIF/MyD88 knockout (TRIF/MyD88−/−) DC. TLR3−/− or TLR7−/− DC were less activated, and reverse transcription was important for the activation of TLR7−/− DC. Moreover, lentivirally transduced DC lacking TLR3 or TLR7 had an impaired capacity to induce antigen-specific CD8+ T-cell responses. In conclusion, we demonstrated TLR-dependent DC activation by lentiviral vectors, explaining their immunogenicity. These data allow the rational development of strategies to manipulate the host's immune response to the transgene.
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Park, Frank. "Lentiviral vectors: are they the future of animal transgenesis?" Physiological Genomics 31, no. 2 (October 2007): 159–73. http://dx.doi.org/10.1152/physiolgenomics.00069.2007.
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Lentiviral vectors have become a promising new tool for the establishment of transgenic animals and the manipulation of the mammalian genome. While conventional microinjection-based methods for transgenesis have been successful in generating small and large transgenic animals, their relatively low transgenic efficiency has opened the door for alternative approaches, including lentiviral vectors. Lentiviral vectors are an appealing tool for transgenesis in part because of their ability to incorporate into genomic DNA with high efficiency, especially in cells that are not actively dividing. Lentiviral vector-mediated transgene expression can also be maintained for long periods of time. Recent studies have documented high efficiencies for lentiviral transgenesis, even in animal species and strains, such as NOD/ scid and C57Bl/6 mouse, that are very difficult to manipulate using the standard transgenic techniques. These advantages of the lentiviral vector system have broadened its use as a gene therapy vector to additional applications that include transgenesis and knockdown functional genetics. This review will address the components of the lentiviral vector system and recent successes in lentiviral transgenesis using both male- and female-derived pluripotent cells. The advantages and disadvantages of lentiviral transgenesis vs. other approaches to produce transgenic animals will be compared with regard to efficiency, the ability to promote persistent transgene expression, and the time necessary to generate a sufficient number of animals for phenotyping.
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Lucke, Susann, Thomas Grunwald, and Klaus Überla. "Reduced Mobilization of Rev-Responsive Element-Deficient Lentiviral Vectors." Journal of Virology 79, no. 14 (July 2005): 9359–62. http://dx.doi.org/10.1128/jvi.79.14.9359-9362.2005.
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ABSTRACT 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 103- to 104-fold lower than for the RRE-containing vector. Thus, RRE-deficient lentiviral vectors provide a novel approach to reduce the risk of vector mobilization.
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Joglekar, Alok V., and Salemiz Sandoval. "Pseudotyped Lentiviral Vectors: One Vector, Many Guises." Human Gene Therapy Methods 28, no. 6 (December 2017): 291–301. http://dx.doi.org/10.1089/hgtb.2017.084.
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Totsugawa, Toshinori, Naoya Kobayashi, Teru Okitsu, Hirofumi Noguchi, Takamasa Watanabe, Toshihisa Matsumura, Masanobu Maruyama, Toshiyoshi Fujiwara, Masakiyo Sakaguchi, and Noriaki Tanaka. "Lentiviral Transfer of the LacZ Gene into Human Endothelial Cells and Human Bone Marrow Mesenchymal Stem Cells." Cell Transplantation 11, no. 5 (July 2002): 481–88. http://dx.doi.org/10.3727/000000002783985620.
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Because one of the attractive characteristics of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors is that it can infect even nondividing cells, a lentivirus-mediated gene delivery system is currently being paid a great deal of attention as an innovative tool for gene transfer into target cells. The purpose of the work was to investigate the efficacy of lentiviral transfer of the LacZ gene into human umbilical vein endothelial cells (HUVECs) and human bone marrow mesenchymal stem cells (HMSCs) in vitro. For the present study, a vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector encoding the E. coli LacZ gene tagged with nuclear localization signal (NLS) was generated in 293T cells by means of the three-plasmid system. The resulting lentiviral vector, LtV-NLS/LacZ, was allowed to infect HUVECs and HMSCs. Approximately 70% of HUVECs were positive for LacZ expression and 50% of HMSCs showed LacZ activity. There was no significant difference in transduction efficacy between early and late-passage phases in both cells. LtV-NLS/LacZ-transduced HUVECs showed gene expression of endothelial markers including CD34 and flt-1 and KDR/flk-1 of vascular endothelial growth factor (VEGF) receptors and had angiogenic potential as efficiently as primarily cultured HUVECs in a Matrigel assay. These findings provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in human endothelial cells and stem cells that could be useful for tissue engineering.
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Lin, Yuan, and Stanton L. Gerson. "Efficient Viral Transduction of Murine Hematopoietic Stem Cells Using HIV-1 Gag-Pol Cross Packaged Simian Immunodeficiency Viral Backbone." Blood 108, no. 11 (November 16, 2006): 5484. http://dx.doi.org/10.1182/blood.v108.11.5484.5484.
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Abstract Lentiviral vectors have been shown to infect non-dividing cells, including hematopoietic stem cell [HSC], and HIV lentiviral vector has been studied extensively in preclinical models. However low HIV lentiviral vector transduction efficiency compared to retroviral vectors, is seen in murine HSC, hampering transplantation and long-term expression of transgene in the recipients. Furthermore, concerns remain regarding the safety of HIV based vectors. Simian Immunodeficiency Viral [SIV] vectors could be safer since the parent virus does not cause disease in humans. However, to model this approach has been difficult because native SIV vectors do not transduce murine cells. We have generated a bicistronic SIV lentiviral SIN vector, containing MGMT and firefly luciferase genes linked by a self-cleavage FMDV 2A sequence. The SIV backbone was kindly provided by Dr. Donald Kohn (University of Southern California). The transgenes are controlled by the MND promoter, which has been shown to express well in murine hematopoietic stem cells. The vector was generated by cross-packaging SIV RNA with HIV-1 ΔR8.91 packaging plasmid and VSVG pseudotyped envelope (Ref. Retrovirology2005, 2:55). Unconcentrated viruses had an average titer of 1E+06 iu/ml, which was similar to HIV-1 lentiviral vectors. In vitro, HIV-1 cross-packaged SIV-mnd-MGMT-2A-Luc vector was able to transduce both human and murine cell lines with no reduction of expression for 10 weeks. In addition, this cross-packaged SIV vector was also able to transduce primary murine bone marrow cells from Balb/C mice with low MOI of 0.5 to 1. Transduced primary murine bone marrow cells maintained transgene expression during a 4 week culture. To analyze in vivo expression, Balb/C bone marrow cells were transduced for 48 hrs in cytokines with the HIV-1 packaged SIV vector and transplanted into irradiated recipients. We used bioluminescent imaging (BLI) to monitor the transgene expression and the dynamic engraftment of transduced murine bone marrow cells. At MOI of 0.5 or 5, transduction efficiencies in murine progenitor cells were 24.4% and 46.7% respectively by PCR of transgene from CFU colonies. Bioluminescent imaging indicated similar engraftment patterns of transduced bone marrow cells by HIV-1 lentiviral vector or cross-packaged SIV lentiviral vector, as early as day 5. Consistent BLI signals indicated sustained expression of transgene in SIV vector transduced bone marrow cells beyond 30 days. With this study, cross-packaged SIV SIN vector could be used as a potential gene transfer vector in both preclinical murine studies and perhaps in clinical trials.
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Paszkiet, Brian, Andrew Worden, Yajin Ni, Saran Bao, Franck Lemiale, Boro Dropulic, and Laurent Humeau. "CD86 and CD54 Co-Expression on VSV-G Pseudotyped HIV-1 Based Vectors Improves Transduction and Activation of Human Primary CD4+ T Lymphocytes." Blood 104, no. 11 (November 16, 2004): 1754. http://dx.doi.org/10.1182/blood.v104.11.1754.1754.
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Abstract We established the first clinical ex vivo HIV-based vector gene therapy trial in humans with HIV+ CD4+ T-cells. Briefly, this therapy involves modifying patient CD4+ T-cells with our modified lentiviral vector carrying an anti-HIV payload. These cells are then activated and expanded, and re-infused back into the patient. However, cGMP regulations require the use of costly clinical grade reagents (i.e. Retronectin™, CD3/CD28 stimulating paramagnetic beads). In an attempt to reduce ex-vivo processing costs, but not at the expense of transduction levels, we sought to determine a way to directly activate CD4+ T-cells with modified lentiviral vectors. 293FT HEK cell lines, used for producing our lentiviral vectors, were modified to co-express the natural CD28 stimulatory ligand B7.2 (CD86) and ICAM-1 (CD54) proteins on their membrane for co-stimulation and anchoring purposes. When CliniMACS purified normal donor CD4+ T cells were co-cultured with CD54/CD86-expressing cells, in the presence of soluble OKT3 CD3 antibody, CD25 and CD69 activation markers were upregulated, indicating that functional proteins were being expressed at the cell membrane. These CD54 and/or CD86 expressing cells could subsequently be transfected with lentiviral vector plasmid constructs in order to produce host-derived CD54 and/or CD86 bearing HIV-based vectors. EGFP-expressing lentiviral vectors, VRX494, with CD54/CD86-modified envelopes were produced both in these cell lines and by transient transfection of all relevant plasmids, and titers were assayed on Hela-Tat cells by FACS. CD54 modified lentiviral vectors showed increased binding to CD4+ T-cells, as evidenced by significant cell clumping. CD86 (as well as CD54 plus CD86) modified lentiviral vector, with soluble OKT3 CD3 antibody, was shown to activate T-cells, above the levels seen with unmodified lentiviral vectors, as evidenced by the increase in cell surface CD25 and CD69 expression and also the increase in cell size. Cellular expansion of modified lentiviral vector transduced CD4+ T cells reached levels close to CD3:CD28 bead stimulated CD4+ T cell controls over a period of 2 to 3 weeks. The CD3/TCR repertoire was assessed by flow cytometry and, compared to the well-established CD3/CD28 coated M450 Dynabeads stimulatory system as a control, no skewing of the repertoire was observed. CD86 was shown to improve levels of transduction in pre-activated lymphocytes with CD3/CD28 coated M450 Dynabeads. However, CD86 co-expression was crucial for transducing minimally activated CD4+ T cells with only soluble OKT3 CD3 antibody. Levels of transduction and activation were on average 2 to 3 times higher with the modified lentiviral vectors. To our knowledge, we are reporting the first generation of lentiviral particles exhibiting an adhesion property with stimulatory abilities. The development of such a lentiviral vector has valuable implications for clinical application by reducing the number of exogenous reagents in large scale cell processing.
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Arsenijevic, Yvan, Adeline Berger, Florian Udry, and Corinne Kostic. "Lentiviral Vectors for Ocular Gene Therapy." Pharmaceutics 14, no. 8 (July 31, 2022): 1605. http://dx.doi.org/10.3390/pharmaceutics14081605.
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This review offers the basics of lentiviral vector technologies, their advantages and pitfalls, and an overview of their use in the field of ophthalmology. First, the description of the global challenges encountered to develop safe and efficient lentiviral recombinant vectors for clinical application is provided. The risks and the measures taken to minimize secondary effects as well as new strategies using these vectors are also discussed. This review then focuses on lentiviral vectors specifically designed for ocular therapy and goes over preclinical and clinical studies describing their safety and efficacy. A therapeutic approach using lentiviral vector-mediated gene therapy is currently being developed for many ocular diseases, e.g., aged-related macular degeneration, retinopathy of prematurity, inherited retinal dystrophies (Leber congenital amaurosis type 2, Stargardt disease, Usher syndrome), glaucoma, and corneal fibrosis or engraftment rejection. In summary, this review shows how lentiviral vectors offer an interesting alternative for gene therapy in all ocular compartments.
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Wang, Yan, Shuai Li, Zhenyu Tian, Jiaqi Sun, Shuobin Liang, Bo Zhang, Lu Bai, et al. "Generation of a caged lentiviral vector through an unnatural amino acid for photo-switchable transduction." Nucleic Acids Research 47, no. 19 (July 30, 2019): e114-e114. http://dx.doi.org/10.1093/nar/gkz659.
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Abstract Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging–uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.
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Dyall, Julie, Jean-Baptiste Latouche, Stefan Schnell, and Michel Sadelain. "Lentivirus-transduced human monocyte-derived dendritic cells efficiently stimulate antigen-specific cytotoxic T lymphocytes." Blood 97, no. 1 (January 1, 2001): 114–21. http://dx.doi.org/10.1182/blood.v97.1.114.
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Abstract Dendritic cells (DCs) are professional antigen-presenting cells that are highly effective adjuvants for immunizing against pathogens and tumor antigens. The potential merit of genetic approaches to loading DCs with antigens is to express high and sustained levels of proteins that can be subsequently processed and presented to T lymphocytes. Replication-defective oncoretroviruses are able to efficiently transduce CD34+ progenitor-derived DCs but not monocyte-derived DCs. Here, it is shown that efficient gene transfer is obtained using a human immunodeficiency virus-1–derived lentiviral vector deleted of all structural and accessory genes. Infection of immature DCs with the lentiviral vector at a multiplicity of infection of 20 resulted in stable gene expression in 30% to 40% of the matured DCs. Proviral DNA was detectable by Alu polymerase chain reaction for the lentiviral but not the oncoretroviral vector. Most importantly, it is demonstrated that lentivirus-transduced DCs were fully functional and effectively activated autologous HLA A2.1+ peripheral blood cytotoxic T lymphocytes (CTLs). DCs expressing lentiviral vector-encoded Flu peptide were at least as efficient as DCs pulsed with the same peptide in stimulating specific CTLs. The efficacy of the lentivirus-transduced DCs was further demonstrated by their ability to directly activate freshly harvested peripheral blood Flu-specific CTLs in the absence of CD4+ T-cell help and exogenous cytokines. The availability of a stable gene delivery system based on a multiply attenuated lentivirus that does not encode any viral protein and that allows sustained antigen presentation by DCs derived from blood monocytes will be very useful for the biologic investigation of DCs and the improvement of immunotherapeutic strategies involving DCs.
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Chinnasamy, Nachimuthu, Michael Milsom, James Neuenfeldt, James Shaffer, Geoffrey P. Margison, Leslie J. Fairbairn, and Dhanalakshmi Chinnasamy. "Development of Novel Multigene Lentiviral Vectors." Blood 104, no. 11 (November 16, 2004): 5272. http://dx.doi.org/10.1182/blood.v104.11.5272.5272.
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Abstract Lentiviral vectors are efficient in transducing dividing and non-dividing cells. Many gene transfer applications require vectors that express more than one protein which include but not limited to therapeutic gene plus a selectable marker gene, multiple genes encoding different subunits of a complex protein or multiple independent genes that cooperate functionally. Previous strategies in constructing multigene lentiviral vectors by other groups included splicing, multiple internal promoters, internal ribosome entry site (IRES), and fusion proteins. In this study we explored the feasibility of expressing homeobox B4 (HOXB4), O6-methylguanine-DNA-methyltransferase (MGMT), and enhanced green fluorescent protein (EGFP) under the control of a single promoter with the use of IRES sequence from encephalomyocarditis (EMCV) virus and/or foot and mouth disease virus (FMDV) 2A cleavage factor in a HIV-1 based self-inactivating lentiviral vector. In bicistronic vectors the small FMDV 2A sequence can be used efficiently as an alternative to IRES. In case of tricistronic vectors the 2A sequence can be used in combination with IRES to obtain simultaneous expression of three cDNAs under the control of a single internal promoter. Monocistronic, bicistronic and tricistronic vectors were constructed and a three-plasmid expression system was used to generate vesicular stomatitis virus glycoprotein pseudotyped lentiviral vector particles. Viral titers were measured by quantification of HIV p24 gag by ELISA. The relative efficiency of transgene expression in transduced cells by various vectors was compared by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. All the multigene vectors produced high titer viral particles and were able to simultaneously express two or three transgenes in transduced cells. However, the expression of EGFP from monocistronic vector as measured by mean fluorescence intensity was 3–8 times and 10–20 times higher than that of bicistronic and tricistronic vectors respectively. Notably expression of second gene encoded by the bicistronic vector containing FMDV 2A was 2–3 fold higher than that of IRES-based vector. Expression of MGMT in monocistronic and bicistronic constructs was also 4 to 20 times higher than tricistronic vectors. The FMDV 2A cleavage factor efficiently mediated the generation of the expected cleavage products from the artificial fusion protein. These vectors can be used in a wide range of applications including the expression of: multiple subunits of a functional protein, tumor antigen and co-stimulatory molecule(s), multiple tumor antigens for immunotherapy applications, and co-expression of selectable marker gene/s with therapeutic gene/s.
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Kim, Yoo-Jin, Yoon-Sang Kim, Andre Larochelle, Gabriel Renaud, Tyra G. Wolfsberg, Rima Adler, Robert E. Donahue, et al. "Sustained high-level polyclonal hematopoietic marking and transgene expression 4 years after autologous transplantation of rhesus macaques with SIV lentiviral vector–transduced CD34+ cells." Blood 113, no. 22 (May 28, 2009): 5434–43. http://dx.doi.org/10.1182/blood-2008-10-185199.
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Abstract We previously reported that lentiviral vectors derived from the simian immunodeficiency virus (SIV) were efficient at transducing rhesus hematopoietic repopulating cells. To evaluate the persistence of vector-containing and -expressing cells long term, and the safety implications of SIV lentiviral vector–mediated gene transfer, we followed 3 rhesus macaques for more than 4 years after transplantation with transduced CD34+ cells. All 3 animals demonstrated significant vector marking and expression of the GFP transgene in T cells, B cells, and granulocytes, with mean GFP+ levels of 6.7% (range, 3.3%-13.0%), 7.4% (4.2%-13.4%), and 5.6% (3.1%-10.5%), respectively. There was no vector silencing in hematopoietic cells over time. Vector insertion site analysis of granulocytes demonstrated sustained highly polyclonal reconstitution, with no evidence for progression to oligoclonality. A significant number of clones were found to contribute at both 1-year and 3- or 4-year time points. No vector integrations were detected in the MDS1/EVI1 region, in contrast to our previous findings with a γ-retroviral vector. These data show that lentiviral vectors can mediate stable and efficient long-term expression in the progeny of transduced hematopoietic stem cells, with an integration profile that may be safer than that of standard Moloney murine leukemia virus (MLV)–derived retroviral vectors.
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Chan, Lucas, Darren Nesbeth, Taylor MacKey, Joanna Galea-Lauri, Joop Gäken, Francisco Martin, Mary Collins, Ghulam Mufti, Farzin Farzaneh, and David Darling. "Conjugation of Lentivirus to Paramagnetic Particles via Nonviral Proteins Allows Efficient Concentration and Infection of Primary Acute Myeloid Leukemia Cells." Journal of Virology 79, no. 20 (October 15, 2005): 13190–94. http://dx.doi.org/10.1128/jvi.79.20.13190-13194.2005.
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ABSTRACT Nonviral producer cell proteins incorporated into retroviral vector surfaces profoundly influence infectivity and in vivo half-life. We report the purification and concentration of lentiviral vectors using these surface proteins as an efficient gene transduction strategy. Biotinylation of these proteins and streptavidin paramagnetic particle concentration enhances titer 400- to 2,500-fold (to 109 CFU/ml for vesicular stomatitis virus G protein and 5 × 108 for amphotropic murine leukemia virus envelope). This method also uses newly introduced membrane proteins (B7.1 and ΔLNGFR) directed to lentiviral surfaces, allowing up to 17,000-fold concentrations. Particle conjugation of lentivirus allows facile manipulation in vitro, resulting in the transduction of 48 to 94% of human acute myeloid leukemia blasts.
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Panigaj, Martin, Michael P. Marino, and Jakob Reiser. "Tagging and Capturing of Lentiviral Vectors Using Short RNAs." International Journal of Molecular Sciences 22, no. 19 (September 23, 2021): 10263. http://dx.doi.org/10.3390/ijms221910263.
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Lentiviral (LV) vectors have emerged as powerful tools for transgene delivery ex vivo but in vivo gene therapy applications involving LV vectors have faced a number of challenges, including the low efficiency of transgene delivery, a lack of tissue specificity, immunogenicity to both the product encoded by the transgene and the vector, and the inactivation of the vector by the human complement cascade. To mitigate these issues, several engineering approaches, involving the covalent modification of vector particles or the incorporation of specific protein domains into the vector’s envelope, have been tested. Short synthetic oligonucleotides, including aptamers bound to the surface of LV vectors, may provide a novel means with which to retarget LV vectors to specific cells and to shield these vectors from neutralization by sera. The purpose of this study was to develop strategies to tether nucleic acid sequences, including short RNA sequences, to LV vector particles in a specific and tight fashion. To bind short RNA sequences to LV vector particles, a bacteriophage lambda N protein-derived RNA binding domain (λN), fused to the measles virus hemagglutinin protein, was used. The λN protein bound RNA sequences bearing a boxB RNA hairpin. To test this approach, we used an RNA aptamer specific to the human epidermal growth factor receptor (EGFR), which was bound to LV vector particles via an RNA scaffold containing a boxB RNA motif. The results obtained confirmed that the EGFR-specific RNA aptamer bound to cells expressing EGFR and that the boxB containing the RNA scaffold was bound specifically to the λN RNA binding domain attached to the vector. These results show that LV vectors can be equipped with nucleic acid sequences to develop improved LV vectors for in vivo applications.
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Liu, Shan-Lu, Christine L. Halbert, and A. Dusty Miller. "Jaagsiekte Sheep Retrovirus Envelope Efficiently Pseudotypes Human Immunodeficiency Virus Type 1-Based Lentiviral Vectors." Journal of Virology 78, no. 5 (March 1, 2004): 2642–47. http://dx.doi.org/10.1128/jvi.78.5.2642-2647.2003.
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ABSTRACT Jaagsiekte sheep retrovirus (JSRV) infects lung epithelial cells in sheep, and oncoretroviral vectors bearing JSRV Env can mediate transduction of human cells, suggesting that such vectors might be useful for lung-directed gene therapy. Here we show that JSRV Env can also efficiently pseudotype a human immunodeficiency virus type 1-based lentiviral vector, a more suitable vector for transduction of slowly dividing lung epithelial cells. We created several chimeric Env proteins that, unlike the parental Env, do not transform rodent fibroblasts but are still capable of pseudotyping lentiviral and oncoretroviral vectors.
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Thrasher, Adrian J. "Progress in Lentiviral Vector Technologies." Human Gene Therapy 24, no. 2 (February 2013): 117–18. http://dx.doi.org/10.1089/hum.2013.2500.
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Gina Vitale. "Lentiviral vector CDMO gets funding." C&EN Global Enterprise 101, no. 5 (February 6, 2023): 13. http://dx.doi.org/10.1021/cen-10105-buscon15.
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Toon, Kamilla, Emma M. Bentley, and Giada Mattiuzzo. "More Than Just Gene Therapy Vectors: Lentiviral Vector Pseudotypes for Serological Investigation." Viruses 13, no. 2 (January 31, 2021): 217. http://dx.doi.org/10.3390/v13020217.
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Serological assays detecting neutralising antibodies are important for determining the immune responses following infection or vaccination and are also often considered a correlate of protection. The target of neutralising antibodies is usually located in the Envelope protein on the viral surface, which mediates cell entry. As such, presentation of the Envelope protein on a lentiviral particle represents a convenient alternative to handling of a potentially high containment virus or for those viruses with no established cell culture system. The flexibility, relative safety and, in most cases, ease of production of lentiviral pseudotypes, have led to their use in serological assays for many applications such as the evaluation of candidate vaccines, screening and characterization of anti-viral therapeutics, and sero-surveillance. Above all, the speed of production of the lentiviral pseudotypes, once the envelope sequence is published, makes them important tools in the response to viral outbreaks, as shown during the COVID-19 pandemic in 2020. In this review, we provide an overview of the landscape of the serological applications of pseudotyped lentiviral vectors, with a brief discussion on their production and batch quality analysis. Finally, we evaluate their role as surrogates for the real virus and possible alternatives.
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Fung, Hua, Andrew E. Sloan, Jane Reese, Basem M. William, Karen Lingas, Lisa R. Rogers, Heather Embree, et al. "Viral Insertion Safety in Patients with Glioblastoma Who Received a Novel Lentiviral MGMT-P140K Gene Therapy to Protect Bone Marrow from Chemotherapy: No Dominant Clonal Evolution Observed with Chemo-Selection." Blood 124, no. 21 (December 6, 2014): 4801. http://dx.doi.org/10.1182/blood.v124.21.4801.4801.
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Abstract INTRODUCTION: To protect normal bone marrow from chemotherapy in glioblastoma patients, we have developed a novel strategy by introducing a strong DNA repair protein, mutant (P140K) of human methylguanine methyltransferase (MGMT), into patients’ CD34+ hematopoietic progenitors (HPC) by lentiviral gene transfer leading to selective expansion of drug-resistant P140K-MGMT CD34+ cells and their myeloid and immune cell progeny. METHODS: To achieve long-term stable expression of the P140K-MGMT gene, we used a lentiviral vector which integrates into the host genome. However, viral insertion mutagenesis has raised safety concerns; as the previous γ-retroviral vectors were associated with insertion mutations leading to development of acute lymphoblastic leukemia in 20% of treated patients. Nevertheless, new improved lentiviral vector with safe feature of insertion site far away from gene transcription start site has been developed. Here we evaluated the safety of a lentivirus vector under selection pressure of chemotherapy. HYPOTHESIS: Our lentiviral vector is safer than traditional γ-retroviral vectors as evident by lack of early clonal dominance even with a chemo-selection. RESULTS: Three glioblastoma patients were recruited and underwent resection, after which CD34+ HPC were mobilized with filgrastim, isolated by magnetic bead separation (Miltneyi CliniMACS), and transduced ex vivo with a GMP-grade lentiviral P140K-MGMT vector (Lentigen Corp). Subsequently, patients received radiation/temozolomide for 6 weeks and up to five cycles of monthly O6-benzylguanine/temozolomide (BG/TMZ) treatment. As a result, all three patients demonstrated a 5-15 fold selective expansion of P140K-MGMT positive HPC and their progeny granulocyte and mononuclear cells in peripheral blood and a small number of CFUs from bone marrow indicating a drug-selection mechanism. The viral insertion sites in the cells of these three patients were closely monitored in each chemotherapy cycle and the patients were followed for up to 1 year after the last therapy. We mapped a total of 17,308 viral insertion sites (VIS), for patient 1(6,146), patient 2(2,081) and patient 3(9,081) and the unique viral insertion sites (UVIS) was 382, i.e. 135, 76 and 171 for patient 1, patient 2 and patient 3 respectively. Overall, during the drug-treatment period, there were no persistent UVIS. Moreover, at the conclusion of BG/TMZ treatment, the VIS number sharply diminished. CONCLUSION: Gene transfer of LV MGMTP140K vector into hematopoietic progenitor cells did not lead to clonal dominance during or after drug selection. Dose escalation of BG/TMZ will define hematopoietic tolerance and treatment response. Disclosures Embree: Lentigen Technology Inc: Employment. Dropulic:Lentigen Technology Inc: Employment, Patents & Royalties.
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Grund, Nadja, Patrick Maier, Uwe Appelt, Heike Allgayer, Frederik Wenz, W. Jens Zeller, Stefan Fruehauf, and Stefanie Laufs. "Impact of Chemoselective Pressure on Integration Site Patterns of Lentivirally Transduced Human Hematopoietic Stem Cells." Blood 112, no. 11 (November 16, 2008): 4622. http://dx.doi.org/10.1182/blood.v112.11.4622.4622.
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Abstract Hematologic side effects of cancer chemotherapy like myelosuppression are frequently dose-limiting. Lentiviral gene therapy with cytostatic drug resistance gene transfer to human hematopoietic stem cells (CD34+) is a promising approach to overcome this problem. In this context it is of interest if chemotherapy mediated selection has an impact on lentiviral integration site patterns of transduced hematopoietic stem cells (CD34+). Concerning this issue, human CD34+ cells transduced with a lentiviral self-inactivating (SIN) vector encoding MGMTP140K (the O6-BG resistant mutant of O6-methylguanine- DNA methyltransferase) were in vitro treated with the alkylating agent BCNU. For integration site analysis LM-PCR was performed and integration patterns of the treated and untreated CD34+ cells were analyzed and compared with an in silico set of 106 random integrations. We found different integration preferences of the lentiviral vector between either the treated (82 integrations) or the untreated (30 integrations) CD34+ cells and the in silico set: both groups showed chromosomal preferences, a significant bias for integrations in genes (74,4% in the treated, respectively 70% in the untreated to 40% in the in silico group), especially by favouring introns, a random integration distribution regarding transcription start sites (TSS), and most importantly no significant differences concerning the number of integrations in or near cancer genes. Concerning all integration characteristics we could not find significant differences when comparing the untreated with the treated group. In conclusion, the general distribution of lentiviral integrations in either untreated or treated human CD34+ cells showed no distinct differences between both groups but significant differences compared to the in silico integration set. These results suggest that chemoselection of cells lentivirally overexpressing a specific chemoresistence gene might not influence the integration pattern. Therefore chemotherapy pressure seems not to hamper the safety of lentiviral vectors in gene transfer studies.
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Persons, Derek A., Phillip W. Hargrove, Esther R. Allay, Hideki Hanawa, and Arthur W. Nienhuis. "The degree of phenotypic correction of murine β-thalassemia intermedia following lentiviral-mediated transfer of a human γ-globin gene is influenced by chromosomal position effects and vector copy number." Blood 101, no. 6 (March 15, 2003): 2175–83. http://dx.doi.org/10.1182/blood-2002-07-2211.
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Increased fetal hemoglobin (HbF) levels diminish the clinical severity of β-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell–targeted gene transfer of a γ-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based γ-globin vectors for expression in transduced erythroid cell lines. Compared with γ-globin, oncoretroviral vectors containing either a β-spectrin or β-globin promoter and the α-globin HS40 element, a γ-globin lentiviral vector utilizing the β-globin promoter and elements from the β-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human γ-globin was observed with levels of vector-encoded γ-globin mRNA ranging from 9% to 19% of total murine α-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of β-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric mα2hγ2molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. γ-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a γ-globin lentiviral vector for gene therapy of β-thalassemia.
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Fichter, Christina, Anupriya Aggarwal, Andrew Kam Ho Wong, Samantha McAllery, Vennila Mathivanan, Bailey Hao, Hugh MacRae, et al. "Modular Lentiviral Vectors for Highly Efficient Transgene Expression in Resting Immune Cells." Viruses 13, no. 6 (June 18, 2021): 1170. http://dx.doi.org/10.3390/v13061170.
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Gene/cell therapies are promising strategies for the many presently incurable diseases. A key step in this process is the efficient delivery of genes and gene-editing enzymes to many cell types that may be resistant to lentiviral vector transduction. Herein we describe tuning of a lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. The motivation for this was to find solutions for HIV gene therapy efforts. Through selection of the optimal viral envelope and further modification to its expression, lentiviral fusogenic delivery into resting CD4+ T cells exceeded 80%, yet Sterile Alpha Motif and HD domain 1 (SAMHD1) dependent and independent intracellular restriction factors within resting T cells then dominate delivery and integration of lentiviral cargo. Overcoming SAMHD1-imposed restrictions, only observed up to 6-fold increase in transduction, with maximal gene delivery and expression of 35%. To test if the biologically limiting steps of lentiviral delivery are reverse transcription and integration, we re-engineered lentiviral vectors to simply express biologically active mRNA to direct transgene expression in the cytoplasm. In this setting, we observed gene expression in up to 65% of resting CD4+ T cells using unconcentrated MS2 lentivirus-like particles (MS2-LVLPs). Taken together, our findings support a gene therapy platform that could be readily used in resting T cell gene editing.
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Agosto, Luis M., Jianqing J. Yu, Megan K. Liszewski, Clifford Baytop, Nikolay Korokhov, Laurent M. Humeau, and Una O'Doherty. "The CXCR4-Tropic Human Immunodeficiency Virus Envelope Promotes More-Efficient Gene Delivery to Resting CD4+ T Cells than the Vesicular Stomatitis Virus Glycoprotein G Envelope." Journal of Virology 83, no. 16 (June 3, 2009): 8153–62. http://dx.doi.org/10.1128/jvi.00220-09.
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ABSTRACT Current gene transfer protocols for resting CD4+ T cells include an activation step to enhance transduction efficiency. This step is performed because it is thought that resting cells are resistant to transduction by lentiviral-based gene therapy vectors. However, activating resting cells prior to transduction alters their physiology, with foreseeable and unforeseeable negative consequences. Thus, it would be desirable to transduce resting CD4+ T cells without activation. We recently demonstrated, contrary to the prevailing belief, that wild-type human immunodeficiency virus (HIV) integrates into resting CD4+ T cells. Based on that finding, we investigated whether a commonly used, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped lentiviral gene therapy vector could also integrate into resting CD4+ T cells. To investigate this, we inoculated resting CD4+ T cells with lentiviral particles that were pseudotyped with VSV-G or CXCR4-tropic HIV Env and assayed binding, fusion, reverse transcription, and integration. We found that the VSV-G-pseudotyped lentiviral vector failed to fuse to resting CD4+ T cells while HIV Env-pseudotyped lentiviral vectors fused, reverse transcribed, and integrated in resting cells. Our findings suggest that HIV Env could be used effectively for the delivery of therapeutic genes to resting CD4+ T cells and suggest that fusion may be the critical step restricting transduction of resting CD4+ T cells by lentiviral gene therapy vectors.
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Hargrove, Phillip W., Steven Kepes, Hideki Hanawa, Cheng Cheng, Geoff Neale, Arthur W. Nienhuis, and Derek A. Persons. "Assessment of Changes in Gene Expression Caused by Insertions of a Globin Lentiviral Vector Containing Globin Regulatory Elements or a Lentiviral Vector Containing Retroviral LTR Elements." Blood 104, no. 11 (November 16, 2004): 497. http://dx.doi.org/10.1182/blood.v104.11.497.497.
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Abstract The development of lymphoid leukemia in two children with X-SCID who underwent gene therapy was partially due to activation of the LMO-2 proto-oncogene by the retroviral LTR of the vector which inserted nearby (Hacein-Bey-Abina et al., Science 2003), highlighting the importance of vector design on the potential to activate genes near vector integration sites. As gene therapy vectors for other blood disorders are evaluated, it seems prudent to assess the safety issues regarding insertion for each particular vector in appropriate pre-clinical models. We have focused on developing γ-globin lentiviral vectors for gene therapy of the hemoglobin disorders and have documented correction of a murine model of β-thalassemia in the absence of observable adverse events (Persons et al., Blood 2003; Hanawa et al., Blood 2004). To more thoroughly evaluate the potential for vector-induced genotoxicity, we have examined whether self-inactivating (SIN) γ-globin lentiviral vectors containing erythroid-specific, β-globin locus enhancer elements can alter the expression of genes nearby the vector insertion site, as the retroviral LTR did in the X-SCID trial. To ascertain whether an integrated globin vector could influence endogenous transcriptional activity in erythroid precursors, 15 clonal spleen colony erythroblast populations (≥ 95% erythroid) containing lentiviral globin vector insertions and 15 untransduced control clones were derived from bone marrow cells of β-thalassemic mice. The transcriptional profile of each clone was determined using the Affymetrix Mouse 430A microarray (representing ~15,000 genes). Expression of 4500–6000 genes was observed in all samples. Ligation-mediated PCR was used to obtain the vector-genomic DNA junction sequences, allowing identification of vector insertion locations in 13 of the clones using the NCBI database. Of these, 6 globin vector clones had 16 genes, including N-ras, which were located within 100kb of the vector insertion site and were represented on the array. Only one gene, D3Jfr1, encoding a “cold shock” DNA binding protein and which was disrupted by an intronic vector insertion, had a change in signal value relative to the mean signal value of the controls. Real time RT-PCR confirmed a 4-fold reduction in expression of this gene. Both microarray and real time RT-PCR demonstrated that expression of N-ras was unchanged. For comparison, 15 clones with insertions of a lentiviral vector containing the MSCV retroviral LTR, were also derived, along with 10 additional mock control clones. We are currently analyzing the expression of some 116 genes that lie within 300kb of the vector insertions, relative to the mean expression level in the 25 mock transduced clones. Additionally, we have expanded analysis of the globin vector clones to evaluate changes in expression of 107 genes located within 300kb of the vector insertions. These data should prove useful to assess whether integrated SIN globin lentiviral vectors containing erythroid-specific regulatory elements have a propensity to alter transcriptional activity in the progeny of genetically modified hematopoietic stem cells, relative to vectors containing viral LTR elements.
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D’Costa, Jenice, Heidi M. Brown, Priya Kundra, Alberta Davis-Warren, and Suresh K. Arya. "Human immunodeficiency virus type 2 lentiviral vectors: packaging signal and splice donor in expression and encapsidation." Journal of General Virology 82, no. 2 (February 1, 2001): 425–34. http://dx.doi.org/10.1099/0022-1317-82-2-425.
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Retroviral vectors provide the means for gene transfer with long-term expression. The lentivirus subgroup of retroviruses, such as human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), possesses a number of regulatory and accessory genes and other special elements. These features can be exploited to design vectors for transducing non-dividing as well as dividing cells with the potential for regulated transgene expression. Encapsidation of the transgene RNA in lentiviral vectors is determined by the leader sequence-based multipartite packaging signal. Embedded in the packaging signal is a major splice donor site that, this study shows, is not by itself essential for transgene expression or encapsidation. We designed HIV-2 vectors that contained all the sequence elements thought to be necessary and sufficient for vector RNA encapsidation. Unexpectedly, despite abundant expression, only a small fraction of the transgene RNA was encapsidated and the titre of the vector was low. Redesign of the vector with a mutant splice donor resulted in increased vector RNA encapsidation and yielded vectors with high titre. Inefficient encapsidation by the conventionally designed vector was not due to suboptimal Rev responsive element (RRE)–Rev function. Varying the length of RRE in the vector did not change vector RNA encapsidation, nor did the introduction of a synthetic intron into the mutant vector. The vector RNA with the intact splice donor may have been excessively spliced, decreasing the amount of packageable RNA. A titre of 105 transducing units (TU)/ml was readily obtained for vectors with the neo or GFP transgene, and the vector could be concentrated to a titre of 1–5×107 TU/ml.
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Sosale, Nisha, Richard K. Tsai, Irena Ivanovska, Philip W. Zoltick, and Dennis E. Discher. "Reducing Immune Response against Lentiviral Vectors: Lentiviral Vector Presentation of CD47, The ‘Marker of Self’." Biophysical Journal 100, no. 3 (February 2011): 403a. http://dx.doi.org/10.1016/j.bpj.2010.12.2393.
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Hanawa, Hideki, Derek A. Persons, and Arthur W. Nienhuis. "Mobilization and Mechanism of Transcription of Integrated Self-Inactivating Lentiviral Vectors." Journal of Virology 79, no. 13 (July 1, 2005): 8410–21. http://dx.doi.org/10.1128/jvi.79.13.8410-8421.2005.
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ABSTRACT Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3′ long terminal repeat (LTR) is copied over into the 5′ LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector.
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Herbst, Friederike, Claudia R. Ball, Francesca Tuorto, Wei Wang, Ulrich Kloz, Frank van der Hoeven, Frank Lyko, Christof Von Kalle, and Hanno Glimm. "Impaired Lentiviral Transgene Expression In Vivo Caused by Massive Methylation of SFFV Promoter Sequences." Blood 116, no. 21 (November 19, 2010): 3760. http://dx.doi.org/10.1182/blood.v116.21.3760.3760.
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Abstract Abstract 3760 Lentiviral vectors (LV) assure stable transgene expression in vivo, allowing to investigate genes and their functions. In recent years, lentiviral gene transfer was considered to facilitate the generation of transgenic mice with a higher yield of transgenic offspring as compared to commonly used DNA microinjection. We applied LV to generate a mouse model transgenic for SETBP1 and eGFP. Murine zygotes were infected at dE0.5 with lentiviral particles directly injected into the perivitelline space. Specific PCRs for either the SETBP1 transgene or for the WPRE element of the lentiviral construct verified complete lentiviral integration in newborn pups (F0). Lentiviral integration sites were detected using highly sensitive LAM-PCR in 65% of 31 analyzed F0 mice. Germline transmission was shown in a total of 33% vector positive offspring from 5 out of 9 F0 mice. However, no ectopic transcription and overexpression of neither SETBP1 nor eGFP could be detected in transgenic mice. We therefore analyzed the methylation status of the internal SFFV promoter (SFFVp) by bisulfite sequencing. Extensive methylation (around 90%) could be assessed in 18 of 18 analyzed CpGs within the promoter region in F0 animals and in all progeny determined (n=12). We transduced mES cells with LV.SFFV.Setbp1.IRES.eGFP or the corresponding eGFP-expressing control vector to exclude transgene effects on epigenetic silencing of SFFVp sequences in self-inactivating LVs. Differentiation of ES cells infected with the transgene vector and SFFV driven control vector led to a 1.8 – 3.5 fold decrease of eGFP expression. To analyze whether methylation of SFFVp sequences is a common event even in adult tissues, we analyzed the methylation status of peripheral blood in mice transplanted with bone marrow cells transduced with either gammaretroviral vectors (RV) or LV 3 months after transplantation (n=7). Interestingly, SFFVp sequences in peripheral blood of mice transplanted with LV transduced bone marrow were stronger methylated than CpGs of SFFVp in RV transplants. Our data demonstrate that the commonly used SFFV promotor is highly methylated with remarkable strength and frequency during development in vivo and differentiation in vitro. We conclude that lentiviral vectors using an internal SFFV promoter are not suitable for the generation of transgenic mice or constitutive expression studies in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.
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Dijon, Marilyne, Caroline Torne-Celer, Cecile Tonnelle, and Christian Chabannon. "Recombination Occurs in Lentiviral Vectors Designed To Express Both EGFP and EYFP in Human Hematopoietic Cells." Blood 104, no. 11 (November 16, 2004): 5249. http://dx.doi.org/10.1182/blood.v104.11.5249.5249.
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Abstract Recombinant lentiviruses are widely used vectors for in vitro and in vivo long-term gene transfer. Many of the initial gene transfer studies were conducted using single reporter genes. However, for many gene delivery applications, expression and detection of two genes are useful. The goal of this study was to explore the feasibility of directing the independent expression of two marker genes from a single HIV-1 derived lentiviral vector. We used the enhanced green fluorescent protein (EGFP) and the enhanced yellow fluorescent protein (EYFP) as markers. Because of spectral overlap of these fluorescent proteins, we modified the optical bench of a FACScalibur flow cytometer (BDIS), with insertion of Omega Optical filters, in order to detect the two emitted fluorescences. To independently co-express the marker genes, we tested two different promoter associations in the same lentiviral backbone. In the first case, the EF1α promoter drove EGFP transcription, and EYFP was translated from spliced RNA transcribed from the LTR promoter. In the second case, two internal promoters : EF1α and CMV were inserted in a SIN (Self Inactivating) vector. Different vectors were generated from the pTRIP deltaU3 EF1α EGFP and pTRIP EF1α EGFP lentiviral vectors (gifts from P. Charneau), with transcriptional units and viral elements in variable positions. Following transduction of hematopoietic and non-hematopoietic cell lines, FACS and PCR analyses allowed to quantify transduction and integration efficiencies, respectively. Addition of EYFP in the original single gene lentiviral vector resulted in a decrease in the overall percentage of fluorescent cells. Moreover, vectors using splice mechanism produced weak co-expression of both genes (less than 4% of double positive cells). However, one of the SIN vector that contains two internal promoters transduced 32 to 100% of Nalm6 human pre-B cells, depending on the MOI; of these fluorescent cells, 12 to 78% were double positive. The size of the provirus was assessed in transduced cells, using southern blotting. Cells transduced with vectors coding for the two marker genes contained DNA sequences with sizes below the expected values; these observations suggest that recombination occurred within the vector sequence during or after transduction. This was further confirmed by southern blotting of sorted single and double positive cells; again, bands with lower than expected sizes were detected. We conclude that two highly homologous sequences can lead to recombination when inserted in close positions into a lentiviral backbone. This conclusion was reinforced by the observation that substitution of the DsRed2 coding sequence to EYFP in the same lentiviral vector that also encodes EGFP produced 100% double positive cells following transduction.
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Mirow, Manuela, Lea Isabell Schwarze, Boris Fehse, and Kristoffer Riecken. "Efficient Pseudotyping of Different Retroviral Vectors Using a Novel, Codon-Optimized Gene for Chimeric GALV Envelope." Viruses 13, no. 8 (July 27, 2021): 1471. http://dx.doi.org/10.3390/v13081471.
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The Gibbon Ape Leukemia Virus envelope protein (GALV-Env) mediates efficient transduction of human cells, particularly primary B and T lymphocytes, and is therefore of great interest in gene therapy. Using internal domains from murine leukemia viruses (MLV), chimeric GALV-Env proteins such as GALV-C4070A were derived, which allow pseudotyping of lentiviral vectors. In order to improve expression efficiency and vector titers, we developed a codon-optimized (co) variant of GALV-C4070A (coGALV-Env). We found that coGALV-Env mediated efficient pseudotyping not only of γ-retroviral and lentiviral vectors, but also α-retroviral vectors. The obtained titers on HEK293T cells were equal to those with the classical GALV-Env, whereas the required plasmid amounts for transient vector production were significantly lower, namely, 20 ng coGALV-Env plasmid per 106 293T producer cells. Importantly, coGALV-Env-pseudotyped γ- and α-retroviral, as well as lentiviral vectors, mediated efficient transduction of primary human T cells. We propose that the novel chimeric coGALV-Env gene will be very useful for the efficient production of high-titer vector preparations, e.g., to equip human T cells with novel specificities using transgenic TCRs or CARs. The considerably lower amount of plasmid needed might also result in a significant cost advantage for good manufacturing practice (GMP) vector production based on transient transfection.
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Buffa, Viviana, Donatella R. M. Negri, Pasqualina Leone, Roberta Bona, Martina Borghi, Ilaria Bacigalupo, Davide Carlei, Cecilia Sgadari, Barbara Ensoli, and Andrea Cara. "A single administration of lentiviral vectors expressing either full-length human immunodeficiency virus 1 (HIV-1)HXB2 Rev/Env or codon-optimized HIV-1JR-FL gp120 generates durable immune responses in mice." Journal of General Virology 87, no. 6 (June 1, 2006): 1625–34. http://dx.doi.org/10.1099/vir.0.81706-0.
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Genetic immunization using viral vectors provides an effective means to elicit antigen-specific cellular immune responses. Several viral vectors have proven efficacious in inducing immune responses after direct injection in vivo. Among them, recombinant, self-inactivating lentiviral vectors are very attractive delivery systems, as they are able to efficiently transduce into and express foreign genes in a wide variety of mammalian cells. A self-inactivating lentiviral vector was evaluated for the delivery of human immunodeficiency virus 1 (HIV-1) envelope sequences in mice in order to elicit specific immune responses. With this aim, BALB/c mice were immunized with a single injection of self-inactivating lentiviral vectors carrying either the full-length HIV-1HXB2 Rev/Env (TY2-IIIBEnv) or the codon-optimized HIV-1JR-FL gp120 (TY2-JREnv) coding sequence. Both vectors were able to elicit specific cellular responses efficiently, as measured by gamma interferon ELISPOT and chromium-release assays, upon in vitro stimulation of splenocytes from BALB/c immunized mice. However, only the TY2-JREnv-immunized mice were able to elicit specific humoral responses, measured as anti-gp120 antibody production. These data provide the first evidence that a single, direct, in vivo administration of a lentiviral vector encoding a viral gene might represent a useful strategy for vaccine development.
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Hanawa, Hideki, Derek A. Persons, Takashi Shimada, and Arthur W. Nienhuis. "Diminished Mobilization of Self-Inactivating (SIN) Lentiviral Vectors Containing Globin Regulatory Elements Compared to Those Containing a Retroviral Long Terminal Repeat." Blood 104, no. 11 (November 16, 2004): 5271. http://dx.doi.org/10.1182/blood.v104.11.5271.5271.
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Abstract Stem cell transfer was successful in treating severe combined immunodeficiency due to deficiencies of the common γ-chain of the lymphoid cytokine receptor (N Engl J Med346:1185, 2002) and adenosine deaminase (Science296:2410, 2002) but 2 of 10 children in the common γ-chain trial developed a lymphoproliferative disease secondary in part due to activation of the LMO2 proto-oncogene by the retroviral long terminal repeat (LTR) (Science302:415, 2003). Both oncoretroviral and lentiviral vectors integrate preferentially into transcriptionally active genes so that vector design to improve safety is important. In focusing on the interactions between vector sequences and the genome surrounding integration sites, we have used self-inactivating (SIN) lentiviral vectors with transcriptionally inactive LTRs. One assay detects mobilization of the vector genome by rescue of a GFP marker while a second detects vector encoded tat transcription (Blood102:249a, 2003). Approximately 1 in 1,000 to 1 in 3,000 vector genomes containing the Mouse Stem Cell Virus (MSCV) LTR are transcribed with transcription arising from cryptic promoters within the vector genome or from nearby genomic sequences. The frequency of genome transcription was diminished by removal of the MSCV LTR enhancer or by addition of an insulator element from the β-globin locus control region (LCR) to the lentiviral SIN LTR. We have now evaluated the effect of globin regulatory elements on transcription of integrated SIN lentiviral vector genomes. Initially, we substituted the LTR GFP cassette in vector MSCV-U3 for one in which elements from the β-globin LCR were linked to the β-globin promoter driving GFP in the reverse transcriptional orientation (d432βGFPim). Two additional vectors were also studied; the globin LCR β-promoter GFP cassette was reversed and the globin RNA processing signals removed to derive vector Fd432βGFP and then it was modified by substituting larger LCR elements to derive vector FmLARβV5GFP. Vector preparations made by co-transfection of 293T cells with vector and packaging plasmids had transducing titers from 1.8 x 107 to 3.0 x 107 TU/ml as assayed on HeLa cells. Three separate polyclonal 293T cell populations transduced 3 times at high vector concentration had copy numbers of the SIN-proviral genomes, as measured by RealTime PCR, that averaged 41 with a range of 23–68. Vector mobilization was evaluated by transfection of these populations with packaging plasmids. Conditioned media harvested from the transfected cells were then assayed for transfer of the GFP marker on HeLa cells. The mobilized titers were normalized based on vector copy number. The mobilized titer of the MSCV-U3 was 38,000 ± 3500 and that of d432βGFPim was only 810 ± 100 (p = 0.0004). The mobilized titers of the vectors in which the globin regulatory elements were in the forward orientation were also significantly lower than MSCV-U3; Fd432βGFP was 2100 ± 250 (p = 0.005) and FmLARβV5GFP was 1600 ± 120 (p = 0.0005). Those data suggest that the globin LCR elements are less likely than the retroviral LTR to induce transcription of the integrated vector genome in nonerythroid cells. Our results combined with ongoing analysis of the influence of vector integration on expression of surrounding genes in separate studies will provide a safety profile of globin lentiviral vectors to guide the development of future clinical protocols.
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Buchschacher, Gary L., and Flossie Wong-Staal. "Development of lentiviral vectors for gene therapy for human diseases." Blood 95, no. 8 (April 15, 2000): 2499–504. http://dx.doi.org/10.1182/blood.v95.8.2499.
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Abstract Retroviral vectors derived from murine retroviruses are being used in several clinical gene therapy trials. Recently, progress has been made in the development of vectors based on the lentivirus genus of retroviruses, which ironically includes a major human pathogen, human immunodeficiency virus (HIV). As these vector systems for clinical gene transfer are developed, it is important to understand the rationale behind their design and development. This article reviews the fundamental features of retrovirus replication and of the elements necessary for development of a retroviral vector system, and it discusses why vector systems based on HIV or other lentiviruses have the potential to become important tools in clinical gene therapy.
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Buchschacher, Gary L., and Flossie Wong-Staal. "Development of lentiviral vectors for gene therapy for human diseases." Blood 95, no. 8 (April 15, 2000): 2499–504. http://dx.doi.org/10.1182/blood.v95.8.2499.008k35_2499_2504.
Full textAbstract:
Retroviral vectors derived from murine retroviruses are being used in several clinical gene therapy trials. Recently, progress has been made in the development of vectors based on the lentivirus genus of retroviruses, which ironically includes a major human pathogen, human immunodeficiency virus (HIV). As these vector systems for clinical gene transfer are developed, it is important to understand the rationale behind their design and development. This article reviews the fundamental features of retrovirus replication and of the elements necessary for development of a retroviral vector system, and it discusses why vector systems based on HIV or other lentiviruses have the potential to become important tools in clinical gene therapy.
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Herzog, Roland W. "Lentiviral vector for hemophilia gene therapy." Blood 103, no. 10 (May 15, 2004): 3609–10. http://dx.doi.org/10.1182/blood-2004-03-0820.
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Colin, Angélique, Mathilde Faideau, Noelle Dufour, Gwennaelle Auregan, Raymonde Hassig, Thibault Andrieu, Emmanuel Brouillet, Philippe Hantraye, Gilles Bonvento, and Nicole Déglon. "Engineered lentiviral vector targeting astrocytesIn vivo." Glia 57, no. 6 (April 15, 2009): 667–79. http://dx.doi.org/10.1002/glia.20795.
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Stripecke, Renata, Angelo A. Cardoso, Karen A. Pepper, Dianne C. Skelton, Xiao-Jin Yu, Leo Mascarenhas, Kenneth I. Weinberg, Lee M. Nadler, and Donald B. Kohn. "Lentiviral vectors for efficient delivery of CD80 and granulocyte-macrophage– colony-stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses." Blood 96, no. 4 (August 15, 2000): 1317–26. http://dx.doi.org/10.1182/blood.v96.4.1317.
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Abstract Cell vaccines engineered to express immunomodulators have shown feasibility in eliminating leukemia in murine models. Vectors for efficient gene delivery to primary human leukemia cells are required to translate this approach to clinical trials. In this study, second-generation lentiviral vectors derived from human immunodeficiency virus 1 were evaluated, with the cytomegalovirus (CMV) promoter driving expression of granulocyte-macrophage–colony-stimulating factor (GM-CSF) and CD80 in separate vectors or in a bicistronic vector. The vectors were pseudotyped with vesicular stomatitis virus G glycoprotein and concentrated to high titers (108-109 infective particles/mL). Human acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia cell lines transduced with the monocistronic pHR-CD80 vector or the bicistronic pHR-GM/CD vector became 75% to 95% CD80 positive (CD80+). More important, transduction of primary human ALL and AML blasts with high-titer lentiviral vectors was consistently successful (40%-95% CD80+). The average amount of GM-CSF secretion by the leukemia cell lines transduced with the pHR-GM-CSF monocistronic vector was 2182.9 pg/106 cells per 24 hours. Secretion was markedly lower with the bicistronic pHR-GM/CD vector (average, 225.7 pg/106 cells per 24 hours). Lower amounts of CMV-driven messenger RNA were detected with the bicistronic vector, which may account for its poor expression of GM-CSF. Primary ALL cells transduced to express CD80 stimulated T-cell proliferation in an autologous mixed lymphocyte reaction. This stimulation was specifically blocked with monoclonal antibodies reactive against CD80 or by recombinant cytotoxic T-lymphocyte antigen 4–immunoglobulin fusion protein. These results show the feasibility of efficiently transducing primary leukemia cells with lentiviral vectors to express immunomodulators to elicit antileukemic immune responses.
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Stripecke, Renata, Angelo A. Cardoso, Karen A. Pepper, Dianne C. Skelton, Xiao-Jin Yu, Leo Mascarenhas, Kenneth I. Weinberg, Lee M. Nadler, and Donald B. Kohn. "Lentiviral vectors for efficient delivery of CD80 and granulocyte-macrophage– colony-stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses." Blood 96, no. 4 (August 15, 2000): 1317–26. http://dx.doi.org/10.1182/blood.v96.4.1317.h8001317_1317_1326.
Full textAbstract:
Cell vaccines engineered to express immunomodulators have shown feasibility in eliminating leukemia in murine models. Vectors for efficient gene delivery to primary human leukemia cells are required to translate this approach to clinical trials. In this study, second-generation lentiviral vectors derived from human immunodeficiency virus 1 were evaluated, with the cytomegalovirus (CMV) promoter driving expression of granulocyte-macrophage–colony-stimulating factor (GM-CSF) and CD80 in separate vectors or in a bicistronic vector. The vectors were pseudotyped with vesicular stomatitis virus G glycoprotein and concentrated to high titers (108-109 infective particles/mL). Human acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia cell lines transduced with the monocistronic pHR-CD80 vector or the bicistronic pHR-GM/CD vector became 75% to 95% CD80 positive (CD80+). More important, transduction of primary human ALL and AML blasts with high-titer lentiviral vectors was consistently successful (40%-95% CD80+). The average amount of GM-CSF secretion by the leukemia cell lines transduced with the pHR-GM-CSF monocistronic vector was 2182.9 pg/106 cells per 24 hours. Secretion was markedly lower with the bicistronic pHR-GM/CD vector (average, 225.7 pg/106 cells per 24 hours). Lower amounts of CMV-driven messenger RNA were detected with the bicistronic vector, which may account for its poor expression of GM-CSF. Primary ALL cells transduced to express CD80 stimulated T-cell proliferation in an autologous mixed lymphocyte reaction. This stimulation was specifically blocked with monoclonal antibodies reactive against CD80 or by recombinant cytotoxic T-lymphocyte antigen 4–immunoglobulin fusion protein. These results show the feasibility of efficiently transducing primary leukemia cells with lentiviral vectors to express immunomodulators to elicit antileukemic immune responses.
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Duvergé, Alexis, and Matteo Negroni. "Pseudotyping Lentiviral Vectors: When the Clothes Make the Virus." Viruses 12, no. 11 (November 16, 2020): 1311. http://dx.doi.org/10.3390/v12111311.
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Delivering transgenes to human cells through transduction with viral vectors constitutes one of the most encouraging approaches in gene therapy. Lentivirus-derived vectors are among the most promising vectors for these approaches. When the genetic modification of the cell must be performed in vivo, efficient specific transduction of the cell targets of the therapy in the absence of off-targeting constitutes the Holy Grail of gene therapy. For viral therapy, this is largely determined by the characteristics of the surface proteins carried by the vector. In this regard, an important property of lentiviral vectors is the possibility of being pseudotyped by envelopes of other viruses, widening the panel of proteins with which they can be armed. Here, we discuss how this is achieved at the molecular level and what the properties and the potentialities of the different envelope proteins that can be used for pseudotyping these vectors are.
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Mikkola, Hanna, Niels-Bjarne Woods, Marketa Sjögren, Hildur Helgadottir, Isao Hamaguchi, Sten-Eirik Jacobsen, Didier Trono, and Stefan Karlsson. "Lentivirus Gene Transfer in Murine Hematopoietic Progenitor Cells Is Compromised by a Delay in Proviral Integration and Results in Transduction Mosaicism and Heterogeneous Gene Expression in Progeny Cells." Journal of Virology 74, no. 24 (December 15, 2000): 11911–18. http://dx.doi.org/10.1128/jvi.74.24.11911-11918.2000.
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ABSTRACT Human immunodeficiency virus type 1-based lentivirus vectors containing the green fluorescent protein (GFP) gene were used to transduce murine Lin− c-kit+ Sca1+primitive hematopoietic progenitor cells. Following transduction, the cells were plated into hematopoietic progenitor cell assays in methylcellulose and the colonies were scored for GFP positivity. After incubation for 20 h, lentivirus vectors transduced 27.3% ± 6.7% of the colonies derived from unstimulated target cells, but transduction was more efficient when the cells were supported with stem cell factor (SCF) alone (42.0% ± 5.5%) or SCF, interleukin-3 (IL-3), and IL-6 (53.3 ± 1.8%) during transduction. The, vesicular stomatitis virus glycoprotein-pseudotyped MGIN oncoretrovirus control vector required IL-3, IL-6, and SCF for significant transduction (39.3 ± 9.4%). Interestingly, only a portion of the progeny cells within the lentivirus-transduced methylcellulose colonies expressed GFP, in contrast to the homogeneous expression in oncoretrovirus-transduced colonies. Secondary plating of the primary GFP+ lentivirus vector-transduced colonies revealed vector PCR+ GFP+ (42%), vector PCR−GFP− (46%), and vector PCR+ GFP−(13%) secondary colonies, indicating true genetic mosaicism with respect to the viral genome in the progeny cells. The degree of vector mosaicism in individual colonies could be reduced by extending the culture time after transduction and before plating into the clonal progenitor cell assay, indicating a delay in the lentiviral integration process. Furthermore, supplementation with exogenous deoxynucleoside triphosphates during transduction decreased mosaicism within the colonies. Although cytokine stimulation during transduction correlates with higher transduction efficiency, rapid cell division after transduction may result in loss of the viral genome in the progeny cells. Therefore, optimal transduction may require activation without promoting intense cell proliferation prior to vector integration.
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Lewis, Brian C., Nachimuthu Chinnasamy, Richard A. Morgan, and Harold E. Varmus. "Development of an Avian Leukosis-Sarcoma Virus Subgroup A Pseudotyped Lentiviral Vector." Journal of Virology 75, no. 19 (October 1, 2001): 9339–44. http://dx.doi.org/10.1128/jvi.75.19.9339-9344.2001.
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ABSTRACT We are using avian leukosis-sarcoma virus (ALSV) vectors to generate mouse tumor models in transgenic mice expressing TVA, the receptor for subgroup A ALSV. Like other classical retroviruses, ALSV requires cell division to establish a provirus after infection of host cells. In contrast, lentiviral vectors are capable of integrating their viral DNA into the genomes of nondividing cells. With the intention of initiating tumorigenesis in resting, TVA-positive cells, we have developed a system for the preparation of a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector, pseudotyped with the envelope protein of ALSV subgroup A (EnvA). The HIV(ALSV-A) vector retains the requirement for TVA on the surface of target cells and can be produced at titers of 5 × 103 infectious units (IU)/ml. By inserting the central polypurine tract (cPPT) from the HIV-1 pol gene and removing the cytoplasmic tail of EnvA, the pseudotype can be produced at titers approaching 105 IU/ml and can be concentrated by ultracentrifugation to titers of 107 IU/ml. HIV(ALSV-A) also infects embryonic fibroblasts derived from transgenic mice in which TVA expression is driven by the β-actin promoter. In addition, this lentivirus pseudotype efficiently infects these fibroblasts after cell cycle arrest, when they are resistant to infection by ALSV vectors. This system may be useful for introducing genes into somatic cells in adult TVA transgenic animals and allows evaluation of the effects of altered gene expression in differentiated cell types in vivo.
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Saedi-Marghmaleki, Mojtaba, Mohammad-Taghi Moradi, Payam Ghasemi-Dehkordi, Leyla Hashemi, and Ali Karimi. "Evaluation of lentiviral vector-based green fluorescent protein expression in human gastric cancer cell line." Journal of Shahrekord University of Medical Sciences 21, no. 5 (October 30, 2019): 204–9. http://dx.doi.org/10.34172/jsums.2019.36.
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Background and aims : Human immunodeficiency virus type 1 (HIV-1) based-lentivirus vector is one of the most promising viral vectors for gene delivery in different cell lines including gastric cell lines. Therefore, the aim of this study was to produce a lentivirus vector for transduction and expression of green fluorescent protein (GFP) in human gastric cancer cell line, AGS. Materials and Methods: In this piece of work, Escherichia coli HB101 was transformed with plasmids psPAX2, pTD, and pMD2.G, following the purification of which their DNA was extracted along with their quantity and quality evaluated to be used in the next experiments. Subsequently, to produce the vector, the packaging cells were transfected with the plasmids and the vector containing supernatant was collected and purified using ultracentrifuge. ELISA was used to confirm the construction of the vector. Fluorescent microscopy and flow cytometry were used to check the expression of GFP in the cell line and to calculate the percentage of GFP expression, respectively. Results: In this study, the results of ELISA confirmed the construction of the plasmid used in this study. AGS cells were infected with viruses produced to detect the viral activity and GFP expression was evaluated by fluorescence microscopy and flow cytometry after 72 hours. Based on the results of flow cytometry, GFP was expressed in over 90% of transduced AGS cells. Conclusion: The results of this study showed that lentiviral vector is a highly efficient vector for expression of GFP gene in AGS cell line.
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Schomber, Tibor, Christian P. Kalberer, Aleksandra Wodnar-Filipowicz, and Radek C. Skoda. "Gene silencing by lentivirus-mediated delivery of siRNA in human CD34+ cells." Blood 103, no. 12 (June 15, 2004): 4511–13. http://dx.doi.org/10.1182/blood-2003-07-2397.
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Abstract To derive an efficient system for gene silencing in human hematopoietic stem cells (HSCs) we modified a lentiviral vector for small interfering RNA (siRNA) delivery. For this purpose, an H1 promoter-driven siRNA expression cassette was introduced into a lentiviral vector, and the p53 mRNA was chosen as a target for siRNA-mediated gene silencing. Using the recombinant lentivirus we infected human cord blood-derived CD34+ cells and obtained a transfection efficiency of up to 50%, as determined by expression of enhanced green fluorescent protein (EGFP). In EGFP-positive long-term culture-initiating cell (LTC-IC)- and colony-forming unit cell (CFU-C)-derived cells, we observed a reduction of p53 mRNA of up to 95%. Importantly, this reduction remained stable during several weeks of cell culture. Furthermore, p53 gene silencing resulted in decreased p21 mRNA levels and reduced the sensitivity of CD34+ cells toward the cytotoxic drug etoposide. Thus, lentiviral delivery of siRNA can allow for efficient and stable gene silencing in human HSCs and will be very valuable for further gene function studies. (Blood. 2004;103:4511-4513)