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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.
Nonclinical Safety Evaluation of Advanced Therapies
Published in Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard, Toxicologic Pathology, 2018
Timothy K. MacLachlan, Kendall S. Frazier, Mercedes Serabian
Over the last several years, a new form of gene therapy has emerged that has, based on its ease of use, changed the face of therapeutic approaches in this area and has significant potential to expand rapidly. The concept of “genome editing” is now possible with the advent of engineering various DNA nucleases to target locations in the genome with high specificity and induce double-strand breaks with the intent of eliminating or restoring the function of an endogenous gene or target the insertion of an exogenous gene (Cox 2015). At this point in time, four major classes of nucleases with the intent of current or eventual therapeutic use exist: 1) meganucleases, 2) zinc finger nucleases (ZFNs), 3) transcription activator–like effector nucleases (TALENs) and 4) CRISPR-associated nuclease Cas9. While the meganuclease, ZFN and TALEN systems, recognize DNA via protein/DNA interactions, Cas9 utilizes what is referred to as a “guide RNA” to direct the nuclease to its target. The ZFN- and TALEN-based technologies have entered into clinical trials, and the meganuclease technology may soon follow (Tebas 2014, Qasim 2017). Given the ease of production of individual or even libraries of such targeting guide RNAs for CRISPR/Cas9, there is a substantial amount of focus on this platform. This field is rapidly evolving, with newer platforms emerging that avoid DNA cleavage and modify nucleotides directly (Komor 2016), thus, it will be important for the toxicologic pathologist to keep track of the progress in this area.
Non-Viral Delivery of Genome-Editing Nucleases for Gene Therapy
Published in Yashwant Pathak, Gene Delivery, 2022
In the current era, gene-editing technologies produce a tremendous effect to cure, treat, or prevent various inherited or acquired diseases. These technologies provide flexibility in localization of correct genes for knockdown or recovery of gene expression, insertion of a therapeutic transgene, or correction of mutations associated with genetic diseases [1]. Gene editing technologies can be divided into four types, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered, regularly interspaced, short palindromic repeat-associated nucleases, such as Cas9 [1, 2]. ZFNs, TALENs, and CRISPR/Cas Zinc-finger nucleases and transcription activator-like effector nucleases (TALENs) are hybrid restriction enzymes composed of a DNA-binding domain and a DNA-cleavage domain based on FokI endonuclease [2] These nucleases introduce precise and specific changes of the genome sequence at any genome locus of interest [2–4] [Figure 12.1]. However, the efficient delivery of these therapeutic genes is challenging. Moreover, the potential therapeutic effect mainly relies on safe and efficient delivery of nucleases into the nucleus [Table 12.1]. Among the various types of nucleases, the CRISPR-associated protein 9 (CRISPR/Cas9) system is considered as a one of the most exciting tools for gene editing. The therapeutic efficacy of CRISPR/Cas 9 system depends on base-pairing between the single-guide RNA (sgRNA) and the target DNA [2, 5]. The nuclease can be delivered mainly by two methods, physical and non-viral based [Table 12.1]. Although physical methods, including electroporation and microinjection, produce high transfection efficiency, low cell viability and cell specific delivery may commonly occur with these delivery systems, they also are also difficult for in vivo application [1–5]. Similarly, viral vectors achieve good performance on delivery of CRISPR/Cas9, but are limited by restricted packaging capacity or unwanted genetic mutations and immunogenicity [5, 6], posing concerns about safety in clinical translation [1–3]. In comparison, nonviral delivery methods via nanoparticles have the potential to overcome many of these limitations, particularly with respect to safety, large loading capacity, and in vivo application [5–8]. The first use of gene editing nucleases in humans was discovered in 2009. Zinc-finger nucleases were delivered to the human CD4 T cells isolated from patients with chronic aviremic HIV infection (NCT00842634) to block the functions of CCR5, a major coreceptor of HIV-1. The resistance of CD4 cells to HIV viruses was achieved by modifying the 11% to 28% of alleles with ZFNs, which showed long persistence after administration to the patients [1, 8].
Common therapeutic advances for Duchenne muscular dystrophy (DMD)
Published in International Journal of Neuroscience, 2021
Arash Salmaninejad, Yousef Jafari Abarghan, Saeed Bozorg Qomi, Hadi Bayat, Meysam Yousefi, Sara Azhdari, Samaneh Talebi, Majid Mojarrad
Meganucleases are an effective approach for correcting DMD frame-shift mutations. Chapdelaine et al. firstly, modeled a DMD frame-shift mutation by integrating a well-characterized meganuclease target site into DMD gene, then, delivered a meganuclease expression plasmid in vitro and in vivo into muscle fibers. The results indicated significant increase at the expression level of dystrophin in muscle cells [134]. Following these progressions, Popplewell et al. designed an engineered meganuclease to replace the mutational hotspot (exons 45–55) with lentiviral donor vector containing exons 45–52 through homology-directed repair (HDR) [135]. Following the emergence of the early meganucleases, artificial zinc-finger endonucleases, was raised to perform targeted engineering of genomes and because of the construction of sequence-specific meganucleases for all possible sequences is costly and time consuming, therefore zinc finger endonucleases-based genome editing was replaced by the meganucleases.
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
The first attempt at gene editing in the DMD gene used meganucleases. Proof of principle was shown by insertion of meganuclease target sites in the middle of a dog microdystrophin plasmid containing a frame-shift mutation that was transfected into 293 FT cells [91]. Overexpression of this meganuclease was able to induce small indels at these target sites, which restored the reading frame and microdystrophin. This same approach was applied later in rag/mdx mouse and human myoblasts where restoration of micro-dystrophin in the muscle fibers and myoblasts was observed [92]. Later a meganuclease was designed to cleave within intron 44 in patient-derived myoblasts, just upstream of the deletion hotspot. Repair matrixes carrying exon 45–52 were co-transduced into patient-derived myoblasts carrying a deletion of exon 45–52. This resulted in the production of full-length dystrophin mRNA [93]. However, an important drawback of meganucleases is that the recognition site has to be introduced in the desired gene. Whilst this does ensure low off-target nuclease activity, first having to genetically modify the target gene has its own risk of off-target effects and is very impractical in a clinical setting.
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
From meganucleases to Cas nucleases, tools for genome editing are becoming more efficient and accurate. As the first generation of genome-editing tools, meganucleases can generate DSBs. However, the long length of recognition motifs limits the application of meganucleases in genome editing [8]. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are improved tools that rely on protein structure modification and optimization for DNA binding domains to achieve genome editing for target genes on certain sites [9,10]. For CRISPR-based genome editing, different cleaving sites on DNA are recognized by the different gRNA sequences. Here we illustrate different genome-editing tools together in Figure 1.