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Immunomodulatory Therapies
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Gene delivery of the receptor is the distinguishing step between TCR-T and CAR-T cell therapies. Peripheral blood T cells are transduced by gamma-retroviral or lentiviral vectors which result in high expression of the introduced receptor in the final cell product. However, with viral vectors there is a concern relating to potential oncogenic gene insertion. Also, this process is presently labour intensive and expensive, although potentially cheaper methodologies based on Crispr/Cas9-technologies are being explored. The latter approach does not require the production of viral vectors and provides a more flexible and cheaper platform for gene transduction. Finally, the transduced T cells are expanded. The overall process is illustrated in Figure 9.7.
Molecular Radiation Biology
Published in Kedar N. Prasad, Handbook of RADIOBIOLOGY, 2020
The availability of plasmid and retroviral vectors carrying specific oncogenes has allowed us to insert exogenous genes into any cell type. The process of gene insertion is often referred to as transfection. The transfection of cells with specific genes can be accomplished by several methods, including electroporation and calcium-phosphate-Co-DNA precipitation techniques. The role of cellular oncogenes or genes in radiosensitivity of mammalian cells can be studied by generating stable transfectants that express high levels of genes under investigation.
In Situ Gene Insertion for Immunotherapy Using Vaccinia Virus Vectors
Published in Eric Wickstrom, Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Edmund C. Lattime, Laurence C. Eisenlohr, Michael J. Mastrangelo
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.
Ex vivo gene therapy for lysosomal storage disorders: future perspectives
Published in Expert Opinion on Biological Therapy, 2023
Edina Poletto, Andrew Oliveira Silva, Ricardo Weinlich, Priscila Keiko Matsumoto Martin, Davi Coe Torres, Roberto Giugliani, Guilherme Baldo
However, to produce optimal results, some important issues still need to be addressed. Some of these issues are related to the availability of patients. For example, due to their low frequency and unspecific symptoms and signs, often LSD patients are diagnosed later in life, when a gene therapy procedure is less likely to produce major benefits. To solve this, early-diagnosis strategies such as newborn screening need to be considered. Other issues are related to the gene therapy procedure per se. In particular, one important point that still needs to be addressed more carefully is genotoxicity and cellular transformation, which has been shown in past gene therapy trials for other conditions, such as X-linked severe combined immunodeficiency (SCID). Even with new techniques that allow gene insertion at a more precise spot, such as genome editing systems, off target effects are still a possibility, and detecting these events when at a very low frequency can be challenging.
CRISPR/Cas: from adaptive immune system in prokaryotes to therapeutic weapon against immune-related diseases
Published in International Reviews of Immunology, 2020
Juan Esteban Garcia-Robledo, María Claudia Barrera, Gabriel J. Tobón
The CRISPR/Cas system emerged during the evolution of bacteria and archaea as a form of adaptive immunity against invasion by bacteriophages and plasmids. Based on Watson-Crick base pairing, CRISPR can be optimized for target gene modification in eukaryotes, including disease model organisms and humans. Indeed, CRISPR/Cas9 offers a simple and inexpensive method for disease modeling, genetic screening, and potentially for disease therapy through gene silencing, gene insertion, stem cell modification, and epigenetic editing. As immunological diseases, including autoimmune disorders, have a strong genetic basis, CRISPR appears to be a potent therapeutic weapon to achieve full remission by personalized medicine. Further insights into autoimmune disorder genetics and CRISPR function are necessary, however, to harness the maximum potential of this genomic engineering tool for the development of personalized therapies against specific diseases.
A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing
Published in mAbs, 2019
Kothai Parthiban, Rajika L. Perera, Maheen Sattar, Yanchao Huang, Sophie Mayle, Edward Masters, Daniel Griffiths, Sachin Surade, Rachael Leah, Michael R. Dyson, John McCafferty
A number of approaches have been described to introduce single antibody genes into each cell, including viral-based systems and transposons.5–8 A disadvantage of these approaches is that single copy integration is controlled by limited infection or transfection, requiring a compromise between library size and single gene insertion. In addition, integration within the genome is random, leading to potential variation in transcription level based on the transcriptional activity of the integration locus. Targeting individual antibody genes to a single locus within the population has the additional advantage of effecting transcriptional normalization across the population. Random integration also introduces the possibility of variable levels of gene silencing within the population.9 To fully realize the potential for antibody display on mammalian cells and other higher eukaryotes, there is a need for a system to create large libraries that combine accurate integration into a pre-defined site with an efficiency that allows construction of large libraries. Site-specific integration of transgenes directed by Flp recombinase using the commercial “Flp-In” system has previously been described.10,11 In direct comparison with the nuclease-directed system presented here, we found the Flp-In system to be deficient (see supplementary section), mirroring the rather limited success both in the original publications and subsequently by others.12 This is likely due to the fact that the Flp-In system is designed for accurate integration in a limited number of clones rather than large library construction. Improved integration efficiencies have been achieved using an alternative recombinase with libraries of 20,000 clones being reported.12