Mutagenic Consequences Of Chemical Reaction with DNA
Philip L. Grover in Chemical Carcinogens and DNA, 2019
If insertion occurs into a gene, the gene may be inactivated. Translocating elements include the mutator bacteriophage Mu, insertion sequences, identifiable only by their inactivating effect, and transposons which carry identifiable genes. Many plasmid drug resistance genes are on transposons so that not only may resistance to a drug be transferred between bacteria on a plasmid, but it may be transferred between plasmids as part of a transposon. This natural genetic engineering is of considerable medical importance, and the mechanism is under active investigation. It does not seem related to conventional recombination. As well as the role of transposons in drug resistance, insertion sequences may be involved in such phenomena as bacterial conjugation and the unstable maize mutation systems examined in the classic studies of McClintock.24
In Situ Gene Insertion for Immunotherapy Using Vaccinia Virus Vectors
Eric Wickstrom in Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Vaccinia virus is highly infectious and will efficiently lyse infected tumor cells, thus directly reducing tumor burden. The wild-type virus alone will trigger a cytokine cascade and recruit helper Τ lymphocytes to the injection site (Mitchison, 1970). The virus has been successfully employed for gene insertion in patients at risk for AIDS (Cooney et al., 1991) and in cancer patients (Hamilton et al., 1994). The large size of the virus allows it to serve as a carrier for multiple genes, if needed. Passenger genes are delivered into, and function in, the cytoplasm of the host cell. The lytic property of the virus, as well as the host anti-vaccinia immune response, will prevent unrestricted (and potentially dangerous) cytokine production. However, as discussed above, we have demonstrated that local production of viral gene products can be sustained by repeated injection of vaccinia.
Vaccinia Virus as a Carrier of Vaccine Antigens
F. Y. Liew in Vaccination Strategies of Tropical Diseases, 2017
The most widely used insertion site is the vaccinia TK gene since this provides a means of selection of recombinant (TK–) viruses. Selective methods are necessary since only 0.1% of the total virus from transfected cells is recombinant. The selection of TK– recombinants requires plaquing on TK– cells in the presence of 5-bromodeoxyuridine (BUdR). This restricts the cell lines that may be used and also causes generation of spontaneous TK– mutants which need to be distinguished from true TK– recombinants. Initially this was done by DNA hybridization using 32P-labeled foreign DNA as a probe,31 but subsequently new plasmid vectors have been designed which aid direct selection of recombinant viruses. These vectors contain two vaccinia promoters, one of which is used to drive expression of the desired foreign gene. The second promoter drives expression of a gene which aids selection of recombinants. For example, β-galactosidase expression can be used to enable visual selection of recombinants, since plaques formed by recombinants turn blue if incubated in the presence of X-gal.44 Expression of selectable genetic markers such as the gene conferring resistance to neomycin46 or the herpes simplex virus (HSV) TK gene in vaccinia TK– mutants also enable rapid selection of recombinant viruses.31,48,54
An overview: CRISPR/Cas-based gene editing for viral vaccine development
Published in Expert Review of Vaccines, 2022
Santosh Bhujbal, Rushikesh Bhujbal, Prabhanjan Giram
CRISPR/Cas9-based gene engineering has seen growing use in vaccinology in recent years for structural and functional research of viral genes, as well as to comprehend virus-host interactions [103]. The insertion of exogenous genes and also the replacement of viral genes in viral pathogens are given in Table 1. To improve insertion effectiveness, researchers have looked into using HDR [115,116] and NHEJ techniques to insert exogenous genes [117,118]. In CRISPR/Cas9 knock-in systems, markers and sensors were frequently utilized to facilitate viral visibility and isolation. An example is a case, a recombinant pseudorabies virus (PRV) culture producing firefly luciferase with extended green fluorescent protein was produced by CRISPR/Cas9 based knock-in and shown to be particularly beneficial for antiviral drug and gRNA analysis [119]. Pure transgenic virus clones can be generated in a single round of assessment with the use of single-cell fluorescence-activated cell sorting. HDR with gRNAs and recipient homologous arms are commonly used for gene replacement and exchange [115].
Promise of gene therapy to treat sickle cell disease
Published in Expert Opinion on Biological Therapy, 2018
Zulema Romero, Mark DeWitt, Mark C. Walters
The rapid recent progress in HSPC lentiviral transduction and transplantation, and the replacement of γ-retroviral vectors by SIN-LV have mitigated insertional genotoxicity. However, when using integrating vectors, the possibility of gene activation and inactivation at insertion sites will be always be a concern, and pre-clinical safety studies that can reliably predict the long-term risk of genotoxicity and leukemic transformation are lacking. Therefore, tracking the clonal reconstitution of the transduced stem cells will be crucial to understand the pattern of activity and stability of these transduced HSPC, which will determine the long-term preservation of the therapeutic outcome [118–123]. Similar concerns exist for gene editing therapy. Eliminating or suppressing off-target mutagenesis, which can lead to undesirable indel formation and chromosomal alterations, is necessary to mitigate the risk of leukemic transformation. A recent report also suggests that the frequency of large deletions and insertions that occur near the on-target site might also be more frequent than previously appreciated and suggests that additional concerns about genotoxicity must be expressly addressed during the clinical translation of this technology [124]. In addition, the possibility of enriching edited HSPCs that are deficient in p53 has been suggested [125,126], which is another safety concern. Finally, for CRISPR/Cas9 specifically, the effects of any pre-existing immunity to Cas9 protein are not understood [127]. All these safety issues must be adequately addressed and overcome before clinical development can proceed.
The potential of gene therapy for mucopolysaccharidosis type I
Published in Expert Opinion on Orphan Drugs, 2020
Luisa Natalia Pimentel Vera, Guilherme Baldo
Other strategies were attempted in parallel throughout the years, as alternatives to viral vectors. Non-viral vectors avoid immunogenicity and also have lower cost of production. Sleeping beauty transposon (SBT) is a non-viral vector that combines some advantages of viruses (capacity of integration) without the need of a viral particle [60]. It is composed of a transposomic region, which is replaced by the transgene to be expressed and a transposase, that generates double-stranded breaks in the DNA and allow the insertion of the transgene [61]. Among its advantages is the fact of (i) not relying on an endogenous DNA repair machinery to integrate into the genome, (ii) the system is not restricted to a cell cycle phase, so it is possible to reach and transpose a wide range of cell types including those with high therapeutic potential and (iii) it allows stable and efficient transgene production once transposed [62,63].
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