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Current Application of CRISPR/Cas9 Gene-Editing Technique to Eradication of HIV/AIDS
Published in Yashwant Pathak, Gene Delivery, 2022
Prachi Pandey, Jayvadan Patel, Samarth Kumar
Currently, HAART remains the key strategy for treatment of HIV-1/AIDS patients within the clinic. It can reduce HIV-1 to an undetectable level and make AIDS a chronic disease. Recently, broadly neutralized antibodies showed promising results but still have an extended path to transfer from bench to bedside. With the development of gene editing technologies, like ZFN, TALEN, and CRISPR/Cas9, more and newer work focuses on using these new strategies to eliminate the virus in patients. ZFN, with the dimensions of ∼1 kb, is simpler to deliver. Nevertheless, the limitation of the target site and high off-target effects make it difficult to be applied within the HIV-1/AIDS gene therapy field. TALEN is more flexible in DNA target design and has lower off target effects compared with ZFN. However, the time-consuming and expensive construction of the recognition site of TALEN for a DNA target hampers the event of this gene editing tool. For CRISPR/Cas9, with more convenient and efficient design of target sites, less laborious vector construction, and limited off target effects, it may be applied quickly in every research field, not only in HIV-1/AIDS therapy.
Nucleic Acids as Therapeutic Targets and Agents
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
However, CRISPR-Cas9 is finding significant use in ex vivo applications such as CAR-T or cancer treatment vaccines where immune cells or cancer cells, respectively, are removed from the patient, genetically modified using CRISPR-Cas-9, before being re-administered to patients (see Chapter 9). At the time of writing, the technology has been successfully applied to primary T cells, and the first ever FDA-approved clinical study using CRISPR/Cas9 to develop CAR-T cells is ongoing and is assessing the safety of these genetically modified cells in humans. There is significant enthusiasm for this approach, as CRISPR/Cas9 is cost effective, relatively integration-free in non-targeted DNA sequences, and has the capacity for affecting multiple genes in a single system. Other gene-editing methods such as the zinc finger nucleases and transcription activator-like effector nucleases (TALENs) have also been used for targeted mutagenesis in CAR-T cell development, but these methods require more extensive engineering and optimization work than those needed for the more simple, robust and low cost RNA-guided mechanism of CRISPR/Cas9.
The science of biotechnology
Published in Ronald P. Evens, Biotechnology, 2020
In this short overview, five methods for gene editing will be defined and briefly outlined: (1) ARCUT, (2) meganucleases, (3) ZFN, (4) TALEN, and (5) CRISPER/Cas. ARCUT is artificial restriction DNA cutter. The DNA cleavage involves a pseudo-complementary peptide nucleic acid that specifies the cleavage site, DNA excision and splicing with ethylenediaminetetraacetic acid and cerium, and DNA ligase to foster DNA attachment at the target site. Meganucleases are large protein enzymes that are many in number and naturally occurring and that excise DNA sequences. They are bound to proteins that assist in specifying DNA cleavage sites. They are limited by also naturally occurring repair processes in cells that can also cause changes in other DNA sites. Zinc finger nucleases (ZFNs) are synthetic programmable combinations of a restriction endonuclease (FokI) and small zinc-ion regulated binding domain proteins, which target triple codons (three nucleic acid sites). FokI nucleases are the DNA cleavage domain only with deletion of the DNA recognition domain. FokI requires homodimerization at the target site in order to cleave DNA, such that two zinc finger molecules are needed to target two nearby DNA sites for DNA cleavage. TALEN is a transcription activator-like effector nuclease, a synthetic construction of a restriction endonuclease (FokI also), bound to a DNA-binding protein domain (TAL effector). The TALEN can bind to single nucleic acids and functions similar to the ZFNs.
Developments in reading frame restoring therapy approaches for Duchenne muscular dystrophy
Published in Expert Opinion on Biological Therapy, 2021
Anne-Fleur E. Schneider, Annemieke Aartsma-Rus
Transcription activator-like effector nucleases (TALENs) offer an alternative. TALE domains consist of a repeat variable residue, which are tandem repeats of 34 amino residues. Each repeat variable residue can recognize only one nucleotide and TALEs suffer a lot less from context-dependency than zinc fingers. The TALEN system was utilized to target exon 51 in DMD patient-derived fibroblasts or immortalized myoblasts cell lines which carried a deletion of exon 48–50. Successful introduction of a small deletion in exon 51 could restore the reading frame and resulted in restored dystrophin expression in myoblasts [96]. TALEN was also used to correct the reading frame in DMD patient-derived induced pluripotent stem cells (iPSCs) that lacked exon 44 [97]. In this scenario, TALEN targeted exon 45 and successfully introduced a 1 bp deletion, restoring the dystrophin reading frame. However, this deletion was present for only 40 of the 229 iPSC clones analyzed, indicating that this process is not efficient.
Genome-wide CRISPR screens for the identification of therapeutic targets for cancer treatment
Published in Expert Opinion on Therapeutic Targets, 2020
Vivian Weiwen Xue, Sze Chuen Cesar Wong, William Chi Shing Cho
TALENs were discovered from Xanthomonas. Each repeat domain of TALE recognizes a single base of DNA, which is the main improvement of TALENs compared to ZFNs in genome editing. TALEN is generated by the artificial chimeric fusion of the Fok I DNA cleaving domain to TALE modules. Compared to ZFNs, TALENs are more powerful tools for generating targeted mutagenesis, but their editing efficiency will be limited by the high ratio of CpG regions in targeted sites in the genome [14]. TALENs are widely used in chimeric antigen receptor (CAR) T cell editing and related cancer immunotherapy. Sachdeva et al. applied TALEN-mediated depletion of granulocyte-macrophage colony-stimulating factor (GM-CSF) in CAR-T cells to prevent the cytokine release syndrome and subsequential secretion of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and IL-8, which aims to minimize the side effects of CART therapy in cancer treatment [15]. Besides, TALENs show potential in the clinical practice of allogeneic CAR-T cancer therapy. It was reported that TALEN-mediated editing for B cell maturation antigen receptor on T cells from healthy donors helps to avoid lymphodepletion and increased antitumor immune responses in multiple myeloma [16]. Additionally, TALEN-mediated genome editing is commonly used to engineer human stem cells for research and personalized medicine [17,18].
New technologies in gene therapy for inducing immune tolerance in hemophilia A
Published in Expert Review of Clinical Immunology, 2018
Chiara Borsotti, Antonia Follenzi
Liver gene therapy is a promising strategy for overcoming two main limits of the actual hemophilia replacement treatment: the short coagulation factor half-life and the inhibitor formation.Gene therapy vectors should carry both transcriptional (e.g. cell specific promoter) and post-transcriptional regulatory elements (e.g. miRTs), useful for enhancing specific coagulation factor production and preventing immune activation.Gene and cell therapies may be combined for obtaining the best result in establishing the long-term expression of the corrected gene and the induction of FVIII tolerance.Both humoral and cytotoxic arms of the immune system need to be regulated for successfully preventing and/or eradicating specific-FVIII immune responses.Genome editing by use of programmable nucleases (e.g. TALEN, CRISPR/Cas9) represents a potential new approach with high potential for correcting gene mutations causing diseases.