scholarly journals The Old and the New: Prospects for Non-Integrating Lentiviral Vector Technology

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1103 ◽  
Author(s):  
Luis Apolonia
Keyword(s):  
Insertional Mutagenesis ◽  
Lentiviral Vector ◽  
Genetic Material ◽  
Lentiviral Vectors ◽  
Cell Types ◽  
Target Cells ◽  
Attachment Sites ◽  
Dividing Cells ◽  
Multiple Cell ◽  
Episomal Dna

Lentiviral vectors have been developed and used in multiple gene and cell therapy applications. One of their main advantages over other vectors is the ability to integrate the genetic material into the genome of the host. However, this can also be a disadvantage as it may lead to insertional mutagenesis. To address this, non-integrating lentiviral vectors (NILVs) were developed. To generate NILVs, it is possible to introduce mutations in the viral enzyme integrase and/or mutations on the viral DNA recognised by integrase (the attachment sites). NILVs are able to stably express transgenes from episomal DNA in non-dividing cells or transiently if the target cells divide. It has been shown that these vectors are able to transduce multiple cell types and tissues. These characteristics make NILVs ideal vectors to use in vaccination and immunotherapies, among other applications. They also open future prospects for NILVs as tools for the delivery of CRISPR/Cas9 components, a recent revolutionary technology now widely used for gene editing and repair.

scholarly journals G100R Mutation within 4070A Murine Leukemia Virus Env Increases Virus Receptor Binding, Kinetics of Entry, and Viral Transduction Efficiency

2003 ◽  
Vol 77 (1) ◽  
pp. 739-743 ◽  
Author(s):  
Chi-Wei Lu ◽  
Lucille O'Reilly ◽  
Monica J. Roth
Keyword(s):  
Viral Entry ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cell Types ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Multiple Cell ◽  
Viral Transduction ◽  
Kinetics Of ◽  
Murine Leukemia

ABSTRACT Passage of 4070A murine leukemia virus (MuLV) in D17 cells resulted in a G-to-R change at position 100 within the VRA of the envelope protein (Env). Compared with 4070A MuLV, virus with the G100R Env displayed enhanced binding on target cells, internalized the virus more rapidly, and increased the overall viral titer in multiple cell types. This provides a direct correlation between binding strength and efficiency of viral entry. Deletion of a His residue at the SU N terminus eliminated the transduction efficiency by the G100R virus, suggesting that the G100R virus maintains the regulatory characteristics of 4070A viral entry. The improved transduction efficiency of G100R Env would be an asset for gene delivery systems.

scholarly journals A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
V. Kalidasan ◽  
Wai Hoe Ng ◽  
Oluwaseun Ayodeji Ishola ◽  
Nithya Ravichantar ◽  
Jun Jie Tan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Delivery ◽  
Delivery System ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Model System ◽  
Selection Marker ◽  
Target Cells ◽  
Primary Cells

AbstractGene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

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 The Utilization of Cell-Penetrating Peptides in the Intracellular Delivery of Viral Nanoparticles

Materials ◽  
2019 ◽  
Vol 12 (17) ◽  
pp. 2671 ◽  
Author(s):  
Jana Váňová ◽  
Alžběta Hejtmánková ◽  
Marie Hubálek Kalbáčová ◽  
Hana Španielová
Keyword(s):  
Genetic Material ◽  
Cell Types ◽  
Cell Penetrating Peptides ◽  
Target Cells ◽  
Current State ◽  
Cargo Delivery ◽  
Viral Particles ◽  
Cell Penetrating ◽  
Viral Nanoparticles ◽  
Immunogenic Properties

Viral particles (VPs) have evolved so as to efficiently enter target cells and to deliver their genetic material. The current state of knowledge allows us to use VPs in the field of biomedicine as nanoparticles that are safe, easy to manipulate, inherently biocompatible, biodegradable, and capable of transporting various cargoes into specific cells. Despite the fact that these virus-based nanoparticles constitute the most common vectors used in clinical practice, the need remains for further improvement in this area. The aim of this review is to discuss the potential for enhancing the efficiency and versatility of VPs via their functionalization with cell-penetrating peptides (CPPs), short peptides that are able to translocate across cellular membranes and to transport various substances with them. The review provides and describes various examples of and means of exploitation of CPPs in order to enhance the delivery of VPs into permissive cells and/or to allow them to enter a broad range of cell types. Moreover, it is possible that CPPs are capable of changing the immunogenic properties of VPs, which could lead to an improvement in their clinical application. The review also discusses strategies aimed at the modification of VPs by CPPs so as to create a useful cargo delivery tool.

Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell–activating polypeptide

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2342-2350 ◽  
Author(s):  
Marielle Maurice ◽  
Els Verhoeyen ◽  
Patrick Salmon ◽  
Didier Trono ◽  
Stephen J. Russell ◽  
...  
Keyword(s):  
T Cell ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Genetic Material ◽  
Lentiviral Vectors ◽  
Target Cells ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Blood Lymphocytes ◽  
Hiv 1

In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1–derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G0 to G1 phases of the cell cycle. Compared to unmodified HIV-1–based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

scholarly journals DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors

2013 ◽  
Vol 42 (4) ◽  
pp. e28-e28 ◽  
Author(s):  
Yujia Cai ◽  
Rasmus O. Bak ◽  
Louise Bechmann Krogh ◽  
Nicklas H. Staunstrup ◽  
Brian Moldt ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
High Efficiency ◽  
Therapeutic Potential ◽  
Genetic Material ◽  
Cell Types ◽  
Protein Transduction ◽  
Dna Transposition ◽  
Dna Transposon ◽  
Transposon Insertion ◽  
Direct Delivery

Abstract DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.

scholarly journals Targeted and non-targeted effects of radiation in mammalian cells: An overview

2021 ◽  
Vol 5 (1) ◽  
pp. 013-019
Author(s):  
Ghosh Rita ◽  
Hansda Surajit
Keyword(s):  
Mammalian Cells ◽  
Bystander Effect ◽  
Therapeutic Potential ◽  
Genetic Material ◽  
Direct Interaction ◽  
Ultra Violet ◽  
Cell Types ◽  
Target Cells ◽  
Effects Of Radiation ◽  
Ionizing Radiations

Radiation of different wavelengths can kill living organisms, although, the mechanism of interactions differs depending on their energies. Understanding the interaction of radiation with living cells is important to assess their harmful effects and also to identify their therapeutic potential. Temporally, this interaction can be broadly divided in three stages – physical, chemical and biological. While radiation can affect all the important macromolecules of the cells, particularly important is the damage to its genetic material, the DNA. The consequences of irradiation include- DNA damage, mutation, cross-linkages with other molecules, chromosomal aberrations and DNA repair leading to altered gene expression and/or cell death. Mutations in DNA can lead to heritable changes and is important for the induction of cancer. While some of these effects are through direct interaction of radiation with the target, radiation can interact with the surrounding environment to result in its indirect actions. The effects of radiation depend not only on the total dose but also on the dose rate, LET etc. and also on the cell types. However, action of radiation on organisms is not restricted to interactions with irradiated cells, i.e. target cells alone; it also exerts non-targeted effects on neighboring unexposed cells to produce productive responses; this is known as bystander effect. The bystander effects of ionizing radiations are well documented and contribute largely to the relapse of cancer and secondary tumors after radiotherapy. Irradiation of cells with non-ionizing Ultra-Violet light also exhibits bystander responses, but such responses are very distinct from that produced by ionizing radiations.

scholarly journals Development of an Avian Leukosis-Sarcoma Virus Subgroup A Pseudotyped Lentiviral Vector

2001 ◽  
Vol 75 (19) ◽  
pp. 9339-9344 ◽  
Author(s):  
Brian C. Lewis ◽  
Nachimuthu Chinnasamy ◽  
Richard A. Morgan ◽  
Harold E. Varmus
Keyword(s):  
Transgenic Mice ◽  
Lentiviral Vector ◽  
Transgenic Animals ◽  
Cell Types ◽  
Host Cells ◽  
Target Cells ◽  
Mouse Tumor ◽  
Sarcoma Virus ◽  
Hiv 1

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.

scholarly journals Antioxidant Regulation of Cell Reprogramming

Antioxidants ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 323 ◽  
Author(s):  
Yuichiro J. Suzuki ◽  
Nataliia V. Shults
Keyword(s):  
Transcription Factors ◽  
New Technologies ◽  
Cell Types ◽  
Cell Reprogramming ◽  
Target Cells ◽  
Direct Reprogramming ◽  
Heart Muscle Cells ◽  
Multiple Cell ◽  
Cell Type Specific ◽  
Induced Pluripotent

Discovery of induced pluripotent stem cells (iPSCs) has revolutionized regeneration biology, providing further mechanistic insights and possible therapeutic applications. The original discovery by Yamanaka and co-workers showed that the expression of four transcription factors in fibroblasts resulted in the generation of iPSCs that can be differentiated into various cell types. This technology should be particularly useful for restoring cells with limited proliferative capacities such as adult heart muscle cells and neurons, in order to treat diseases affecting these cell types. More recently, iPSCs-mediated cell reprogramming has advanced to new technologies including direct reprogramming and pharmacological reprogramming. Direct reprogramming allows for the conversion of fibroblasts into cardiomyocytes, neurons or other cells by expressing multiple cell type-specific transcription factors without going through the production of iPSCs. Both iPSC-mediated reprogramming as well as direct reprogramming can also be promoted by a combination of small molecules, opening up a possibility for pharmacological therapies to induce cell reprogramming. However, all of these processes have been shown to be affected by reactive oxygen species that reduce the efficacies of reprogramming fibroblasts into iPSCs, differentiating iPSCs into target cells, as well as direct reprogramming. Accordingly, antioxidants have been shown to support these reprogramming processes and this review article summarizes these findings. It should be noted however, that the actions of antioxidants to support cell reprogramming may be through their ROS inhibiting abilities, but could also be due to mechanisms that are independent of classical antioxidant actions.

scholarly journals Retroviral and Lentiviral Vectors for the Induction of Immunological Tolerance

Scientifica ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Inès Dufait ◽  
Therese Liechtenstein ◽  
Alessio Lanna ◽  
Christopher Bricogne ◽  
Roberta Laranga ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Immunological Tolerance ◽  
Target Cells ◽  
Intracellular Signalling Pathways ◽  
Tolerogenic Dendritic Cells ◽  
Antigen Presenting ◽  
Immunosuppressive Cytokines

Retroviral and lentiviral vectors have proven to be particularly efficient systems to deliver genes of interest into target cells, either in vivo or in cell cultures. They have been used for some time for gene therapy and the development of gene vaccines. Recently retroviral and lentiviral vectors have been used to generate tolerogenic dendritic cells, key professional antigen presenting cells that regulate immune responses. Thus, three main approaches have been undertaken to induce immunological tolerance; delivery of potent immunosuppressive cytokines and other molecules, modification of intracellular signalling pathways in dendritic cells, and de-targeting transgene expression from dendritic cells using microRNA technology. In this review we briefly describe retroviral and lentiviral vector biology, and their application to induce immunological tolerance.

Sign in / Sign up

Export Citation Format

Share Document