China
Africa
Explore chapters and articles related to this topic
Advances in Genome Editing
Published in Yashwant Pathak, Gene Delivery, 2022
The TALEN (Transcription Activator-Like Effector Nucleases) tool was introduced in 2011 to improve the efficiency, reliability, and accessibility of genome editing. The transcription activator-like effectors (TALES) generated by the phytopathogenic bacteria Xanthomonas genus gave rise to the TALEN system (Moore et al., 2014). The activator proteins are members of the DNA binding protein family and, like transcription factors in eukaryotic genomes, can be utilized to promote the expression of their target genes. The TALE proteins’ DNA binding domain is constituted of monomers of 34 amino acid residues, each of which binds one nucleotide in the target nucleotide sequence (Christian et al., 2012). Apart from two hypervariable amino acids (the repeat variable di-residues) at positions 12 and 13, the amino acid sequence of each repeat is fundamentally similar and in charge of identifying a specific nucleotide. (Mak et al., 2013). To produce TALEN-induced targeted genomic alteration, the functional endonuclease FokI is artificially coupled to DNA binding domains to form site-specific DSBs and encourage DNA recombination. The FokI cleavage domain must be dimerized in order to cleave the two strands of the targeted DNA. As a result, TALEN modules are built in pairs to engage opposing DNA target loci, with sufficient separation (12–20 bp) between the two binding sites (Li et al., 2011).
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.
Genetic modification and preconditioning strategies to enhance functionality of mesenchymal stromal cells: a clinical perspective
Published in Expert Opinion on Biological Therapy, 2023
Kasra Moeinabadi-Bidgoli, Radman Mazloomnejad, Alireza Beheshti Maal, Hamid Asadzadeh Aghdaei, Mandana Kazem Arki, Nikoo Hossein-Khannazer, Massoud Vosough
In the case of the genetic modification of MSCs, despite the improvement of different cell characteristics such as migration and residence in the target tissues, promoted cellular replacement repair, as well as general paracrine and endocrine activities of MSCs, many obstacles have hindered final clinical outcomes [186]. The possibility of immunogenicity, the risk of tumorigenesis of viral vectors, and the lower transmission efficiency of non-viral vectors are among these limitations [168,171]. Hence, considering the efficacy of modified MSCs and their side effects including the severity of host immune responses influenced by various factors such as vector dose, route of administration, transgenic nature, and host-related factors, more investigations should be done in order to optimize these factors. In this regard, utilization of novel genome editing technologies such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins 9 (Cas9) nuclease system, which have emerged in recent years, can be useful [187]. Considering the characteristics of these methods as well as their improved results in in vitro and in vivo studies, it is hoped that the use of these methods will improve the results of clinical trials in this field as well.
Approaches for refining and furthering the development of CAR-based T cell therapies for solid malignancies
Published in Expert Opinion on Drug Discovery, 2021
GvHD can alternatively be prevented by eliminating TCR expression in CAR T cells. The TCR α chain is commonly targeted and proof of principle studies have used a variety of technologies to achieve this [129,130]. Transcription activator-like effector nucleases (TALEN) technology can be multiplexed to knock out both the TCR α chain and CD52, which allowed the transferred CAR T cells to be rendered resistant to lymphodepletion regimens [131]. In an elegant study using CRISPR/cas9 technology, the expression of the TCR α chain was simultaneously removed as the CAR was inserted into the genome. By targeting the CAR construct to the TCR α locus, not only was CAR expression simultaneously achieved and regulated as if it were a TCR chain but issues around safety due to insertional mutagenesis were avoided [132,133].
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.