Lentiviral vectors: are they the future of animal transgenesis?

2007 ◽  
Vol 31 (2) ◽  
pp. 159-173 ◽  
Author(s):  
Frank Park
Keyword(s):  
Transgene Expression ◽  
High Efficiency ◽  
Lentiviral Vector ◽  
Transgenic Animals ◽  
Lentiviral Vectors ◽  
Vector System ◽  
Gene Therapy Vector ◽  
Advantages And Disadvantages ◽  
Lentiviral Transgenesis ◽  
Alternative Approaches

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.

Lentiviral vectors: basic to translational

2012 ◽  
Vol 443 (3) ◽  
pp. 603-618 ◽  
Author(s):  
Toshie Sakuma ◽  
Michael A. Barry ◽  
Yasuhiro Ikeda
Keyword(s):  
Gene Delivery ◽  
Lentiviral Vector ◽  
Integration Site ◽  
Marker Gene ◽  
Transgenic Animals ◽  
Lentiviral Vectors ◽  
Cell Types ◽  
Rapid Expansion ◽  
Vector System

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.

scholarly journals Toward Tightly Tuned Gene Expression Following Lentiviral Vector Transduction

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1427
Author(s):  
Audrey Page ◽  
Floriane Fusil ◽  
François-Loïc Cosset
Keyword(s):  
Gene Expression ◽  
Transgene Expression ◽  
Lentiviral Vector ◽  
Basic Research ◽  
Lentiviral Vectors ◽  
Retroviral Vectors ◽  
Clinical Applications ◽  
Long Terminal Repeats ◽  
Vector Systems ◽  
Gene Excision

Lentiviral vectors are versatile tools for gene delivery purposes. While in the earlier versions of retroviral vectors, transgene expression was controlled by the long terminal repeats (LTRs), the latter generations of vectors, including those derived from lentiviruses, incorporate internal constitutive or regulated promoters in order to regulate transgene expression. This allows to temporally and/or quantitatively control transgene expression, which is required for many applications such as for clinical applications, when transgene expression is required in specific tissues and at a specific timing. Here we review the main systems that have been developed for transgene regulated expression following lentiviral gene transfer. First, the induction of gene expression can be triggered either by external or by internal cues. Indeed, these regulated vector systems may harbor promoters inducible by exogenous stimuli, such as small molecules (e.g., antibiotics) or temperature variations, offering the possibility to tune rapidly transgene expression in case of adverse events. Second, expression can be indirectly adjusted by playing on inserted sequence copies, for instance by gene excision. Finally, synthetic networks can be developed to sense specific endogenous signals and trigger defined responses after information processing. Regulatable lentiviral vectors (LV)-mediated transgene expression systems have been widely used in basic research to uncover gene functions or to temporally reprogram cells. Clinical applications are also under development to induce therapeutic molecule secretion or to implement safety switches. Such regulatable approaches are currently focusing much attention and will benefit from the development of other technologies in order to launch autonomously controlled systems.

scholarly journals Adeno-associated virus Type 2 Rep proteins mediate integration of lentiviral vectors

2014 ◽  
Author(s):  
Victor J McAlister ◽  
Anthony T Craig ◽  
Roland A Owens
Keyword(s):  
Virus Type ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Specific Pcr ◽  
Rep Proteins ◽  
Dividing Cells ◽  
Lentivirus Vectors

Aims: Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that is being developed as a gene therapy vector. In dividing cells, AAV2 DNA persists by integration into the host chromosomes. AAV2 is unique among mammalian viruses in its ability to integrate preferentially into a particular locus within human chromosome 19, designated AAVS1(also known as Mbs 85). The AAV2 Rep68 and Rep78 proteins mediate this integration. Recent data suggest that Rep68 and Rep78 can mediate integration of non-AAV2 DNA with free ends. To test this hypothesis, we targeted insertion of different lentiviral vectors to AAVS1. Methods: Cells were co-infected with wild-type AAV2, and integrase-proficient or integrase-deficient lentivirus vectors. A highly specific PCR-based assay was used to detect lentivirus integration at AAVS1. Similar experiments were performed using lentiviral vectors containing the AAV2 rep gene. Results: All lentiviral vectors tested integrated at AAVS1, if the rep gene was present either within the lentiviral vector or supplied in trans. All that was required for integration at AAVS1 was the amino acid sequence shared between Rep68 and Rep78. The results were similar with integrase-proficient or integrase-deficient lentiviral vectors. Conclusions. The inclusion of the rep gene with lentiviral vectors may produce more predictable integration patterns.

scholarly journals Adeno-associated virus Type 2 Rep proteins mediate integration of lentiviral vectors

2014 ◽  
Author(s):  
Victor J McAlister ◽  
Anthony T Craig ◽  
Roland A Owens
Keyword(s):  
Virus Type ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Specific Pcr ◽  
Rep Proteins ◽  
Dividing Cells ◽  
Lentivirus Vectors

Aims: Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that is being developed as a gene therapy vector. In dividing cells, AAV2 DNA persists by integration into the host chromosomes. AAV2 is unique among mammalian viruses in its ability to integrate preferentially into a particular locus within human chromosome 19, designated AAVS1(also known as Mbs 85). The AAV2 Rep68 and Rep78 proteins mediate this integration. Recent data suggest that Rep68 and Rep78 can mediate integration of non-AAV2 DNA with free ends. To test this hypothesis, we targeted insertion of different lentiviral vectors to AAVS1. Methods: Cells were co-infected with wild-type AAV2, and integrase-proficient or integrase-deficient lentivirus vectors. A highly specific PCR-based assay was used to detect lentivirus integration at AAVS1. Similar experiments were performed using lentiviral vectors containing the AAV2 rep gene. Results: All lentiviral vectors tested integrated at AAVS1, if the rep gene was present either within the lentiviral vector or supplied in trans. All that was required for integration at AAVS1 was the amino acid sequence shared between Rep68 and Rep78. The results were similar with integrase-proficient or integrase-deficient lentiviral vectors. Conclusions: The inclusion of the rep gene with lentiviral vectors may produce more predictable integration patterns.

Development of An HIV1-Based Lentiviral Vector Able to Transduce Both Human and Rhesus Blood Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2353-2353
Author(s):  
Naoya Uchida ◽  
Kareem Washington ◽  
Jun Hayakawa ◽  
Matthew Hsieh ◽  
Aylin C. Bonifacino ◽  
...  
Keyword(s):  
Blood Cell ◽  
Cell Lines ◽  
Blood Cells ◽  
Transgene Expression ◽  
Lentiviral Vector ◽  
Vector System ◽  
Transduction Efficiency ◽  
Large Animal ◽  
Preclinical Testing ◽  
Cd34 Cells

Abstract Although the successful application of gene transfer to hematopoietic stem cells using HIV based vectors for the hemoglobinopathies and thalassemias has recently been achieved in rodents, concerns regarding possible side effects, including insertional mutagenesis, mandate large animal preclinical testing. HIV vectors transduce rhesus blood cells poorly due to a species specific block by TRIM5a and APOBEC3G which target HIV capsid proteins (CAs) and viral infectivity factor (Vif), respectively. We developed a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV and SIV by including SIV CA and SIV Vif. Titers of HIV vectors prepared with SIV Vif (8.4±0.69x106 IU/ml, p=0.73) or SIV CA (delta-HIV 8.1±1.1x106 IU/ml, p=0.73) were comparable to that of a normal HIV vector (9.2±0.46x106 IU/ml), while titers of chimeric HIV vectors that included SIV gag-pol or rev-tat were dramatically decreased (0.18-4.5x105 IU/ml, p<0.001). Unlike the HIV chimeric vectors, chimeric SIV vectors that included both HIV gag-pol and rev-tat showed relatively high titers when compared to normal SIV vectors (p=0.88), while titers of chimeric SIV vectors that included HIV CA were very low (p<0.01). To evaluate whether the candidate chimeric HIV vectors were able to transduce both human and rhesus blood cell lines, we performed dose-escalation transductions using the chimeric vectors. The delta-HIV vector showed superior transduction of human blood cell lines (61±2.8% at MOI=3, p<0.01) than the SIV vector (49±1.8%) and could efficiently transduce rhesus blood cell lines (18±0.90% at MOI=7, p<0.001) while the HIV vector could not (1.8±0.05%). To evaluate the transduction efficiency of delta-HIV on human and rhesus hematopoietic cells, we transduced human and rhesus CD34+ cells with delta-HIV and HIV vectors at various MOIs (0.5–50). In human CD34+ cells, the delta-HIV and HIV vectors showed similar transduction efficiency at lower MOIs (51±1.3%, 47±1.7% at MOI=5, p=0.18, respectively), while in rhesus cells, the transduction rates of delta-HIV (48±1.4% at MOI=2) were significantly superior to that of the HIV vector (6.5±0.36% at all MOIs, p<0.01). Among rhesus CFU, the delta-HIV vector produced a significantly higher percentage of GFP positive clones in comparison to those transduced with the HIV vector (Erythroid (E): 77±1.0% vs. 15±2.7% p<0.01, Myeloid (M): 46±1.8% vs. 1.2±0.59% p<0.01, respectively). On the other hand, among human CFU, similar rates of transgene expressing clones were observed (E: 79±0.79% vs. 72±0.64% p<0.01, M: 34±2.0% vs. 37±0.88% p=0.22, respectively). In erythroid and myeloid liquid cultures derived from transduced CD34+ cells, rhesus cells showed higher rates of transgene expression after delta-HIV transduction than after HIV transduction (E: 51±6.4% vs. 11±0.24% p<0.001, M: 27±2.5% vs. 6.1±2.0% p<0.001, respectively). In human erythroid and myeloid cells, the transgene expression rates were similar (E: 66±4.0% vs. 68±2.3% p=0.61, M: 36±1.5% vs. 37±1.5% p=0.93, respectively). In summary, we have developed an HIV-based lentiviral vector system capable of efficient transduction of both human and rhesus blood cells. This vector system should allow preclinical testing of HIV based therapeutic vectors in the large animal model. Figure Figure

scholarly journals Design and Characterization of an “All-in-One” Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 375 ◽  
Author(s):  
Katharina Zimmermann ◽  
Johannes Kuehle ◽  
Anna Christina Dragon ◽  
Melanie Galla ◽  
Christina Kloth ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Transgene Expression ◽  
Cell Activation ◽  
Lentiviral Vector ◽  
Tumor Therapy ◽  
Vector System ◽  
Target Cells ◽  
Efficient Production ◽  
Car T

Genetically modified T cells expressing chimeric antigen receptors (CARs) so far have mostly failed in the treatment of solid tumors owing to a number of limitations, including an immunosuppressive tumor microenvironment and insufficient CAR T cell activation and persistence. Next-generation approaches using CAR T cells that secrete transgenic immunomodulatory cytokines upon CAR signaling, known as TRUCKs (“T cells redirected for universal cytokine-mediated killing”), are currently being explored. As TRUCKs were engineered by the transduction of T cells with two separate vectors, we developed a lentiviral modular “all-in-one” vector system that combines constitutive CAR expression and inducible nuclear factor of activated T cells (NFAT)-driven transgene expression for more efficient production of TRUCKs. Activation of the GD2-specific CAR via GD2+ target cells induced NFAT promoter-driven cytokine release in primary human T cells, and indicated a tight linkage of CAR-specific activation and transgene expression that was further improved by a modified NFATsyn promoter. As proof-of-concept, we showed that T cells containing the “all-in-one” vector system secrete the immunomodulatory cytokines interleukin (IL)12 or IL18 upon co-cultivation with primary human GD2+ tumor cells, resulting in enhanced effector cell properties and increased monocyte recruitment. This highlights the potential of our system to simplify application of TRUCK-modified T cells in solid tumor therapy.

scholarly journals Adeno-associated virus Type 2 Rep proteins mediate integration of lentiviral vectors

2014 ◽  
Author(s):  
Victor J McAlister ◽  
Anthony T Craig ◽  
Roland A Owens
Keyword(s):  
Virus Type ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Specific Pcr ◽  
Rep Proteins ◽  
Dividing Cells ◽  
Lentivirus Vectors

Aims: Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that is being developed as a gene therapy vector. In dividing cells, AAV2 DNA persists by integration into the host chromosomes. AAV2 is unique among mammalian viruses in its ability to integrate preferentially into a particular locus within human chromosome 19, designated AAVS1(also known as Mbs 85). The AAV2 Rep68 and Rep78 proteins mediate this integration. Recent data suggest that Rep68 and Rep78 can mediate integration of non-AAV2 DNA with free ends. To test this hypothesis, we targeted insertion of different lentiviral vectors to AAVS1. Methods: Cells were co-infected with wild-type AAV2, and integrase-proficient or integrase-deficient lentivirus vectors. A highly specific PCR-based assay was used to detect lentivirus integration at AAVS1. Similar experiments were performed using lentiviral vectors containing the AAV2 rep gene. Results: All lentiviral vectors tested integrated at AAVS1, if the rep gene was present either within the lentiviral vector or supplied in trans. All that was required for integration at AAVS1 was the amino acid sequence shared between Rep68 and Rep78. The results were similar with integrase-proficient or integrase-deficient lentiviral vectors. Conclusions: The inclusion of the rep gene with lentiviral vectors may produce more predictable integration patterns.

scholarly journals Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

2015 ◽  
Vol 12 (5) ◽  
pp. 407-415 ◽  
Author(s):  
Yingying Mao ◽  
Renhe Yan ◽  
Andrew Li ◽  
Yanling Zhang ◽  
Jinlong Li ◽  
...  
Keyword(s):  
Transgene Expression ◽  
High Efficiency ◽  
Lentiviral Vectors

scholarly journals Adeno-associated virus Type 2 Rep proteins mediate integration of lentiviral vectors

2014 ◽  
Author(s):  
Victor J McAlister ◽  
Anthony T Craig ◽  
Roland A Owens
Keyword(s):  
Virus Type ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Specific Pcr ◽  
Rep Proteins ◽  
Dividing Cells ◽  
Lentivirus Vectors

Aims: Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that is being developed as a gene therapy vector. In dividing cells, AAV2 DNA persists by integration into the host chromosomes. AAV2 is unique among mammalian viruses in its ability to integrate preferentially into a particular locus within human chromosome 19, designated AAVS1(also known as Mbs 85). The AAV2 Rep68 and Rep78 proteins mediate this integration. Recent data suggest that Rep68 and Rep78 can mediate integration of non-AAV2 DNA with free ends. To test this hypothesis, we targeted insertion of different lentiviral vectors to AAVS1. Methods: Cells were co-infected with wild-type AAV2, and integrase-proficient or integrase-deficient lentivirus vectors. A highly specific PCR-based assay was used to detect lentivirus integration at AAVS1. Similar experiments were performed using lentiviral vectors containing the AAV2 rep gene. Results: All lentiviral vectors tested integrated at AAVS1, if the rep gene was present either within the lentiviral vector or supplied in trans. All that was required for integration at AAVS1 was the amino acid sequence shared between Rep68 and Rep78. The results were similar with integrase-proficient or integrase-deficient lentiviral vectors. Conclusions: The inclusion of the rep gene with lentiviral vectors may produce more predictable integration patterns.

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