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Genome Editing and Gene Therapies: Complex and Expensive Drugs
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2020
In 2015, Liang et al. reported CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes; they found that this genome editing technique could cleave the endogenous ß-globin gene (HBB) but with a poor efficiency of homologous recombination directed repair (HDR) of HBB; moreover, the edited embryos were mosaic and off-target cleavage was detected, too. See also the first study using base editor system to correct disease mutant in human embryos, published by Liang et al. (2017), the paper on gene-editing technologies in embryos in connection with human reproduction (Eguizabal et al., 2019) or the comments of the editor of the Journal Molecular Genetics and Genomics (Hohmann, 2017) on the publication of Tang and colleagues (2017) about the application of CRISPR/Cas-mediated corrections of point mutations in the HBB and G6PD genes in human zygotes. Although the authors used non-viable tripronuclear embryos for their study, this first publication about genome editing in human embryos triggered off a worldwide discussion (among scientists and scientific organizations, regulatory authorities, but also in the general public) about ethical and societal issues related to this new gene therapeutic tool, and genome editing in general.
Nonviral Therapeutic Approaches for Modulation of Gene Expression: Nanotechnological Strategies to Overcome Biological Challenges
Published in Ana Rute Neves, Salette Reis, Nanoparticles in Life Sciences and Biomedicine, 2018
Ana M. Cardoso, Ana L. Cardoso, Maria C. Pedroso de Lima, Amalia S. Jurado
The major disadvantage of these genome editing techniques remains their imperfect specificity, which can pose a risk to the patient as severe as that observed in X-SCID patients treated with retroviral-based gene therapy. In fact, in a study aiming to correct the human hemoglobin p (HBB) and the C-C chemokine receptor type 5 (CCR5) mutated genes, several guide RNA strands were designed to assess the double strand break (DSB) efficiency and specificity of the CRISPR/Cas9 system. After cell transfection with each CRISPR construct, genomic DNA was harvested and sequenced to evaluate the on- and off-target effects of each guide strand. This study demonstrated that genes with high homology, such as those presented by the human hemoglobin S (HBD) and the C-C chemokine receptor type 2 (CCR2), with respect to HBB and CCR5, respectively, suffered an increased risk of off-target effects, which can be as small as a point mutation or as large as the inversion of a chromosome portion [91]. Thus, cautious evaluation of the off-target effects is necessary to guarantee the safety of the CRISPR/Cas system for a given target.
CRISPR-Based Genome Engineering in Human Stem Cells
Published in Deepak A. Lamba, Patient-Specific Stem Cells, 2017
Thelma Garcia, Deepak A. Lamba
One of the first reports on the feasibility of CRISPR technology to generate disease models in hiPSC lines was described by the Ellerby group for studying Huntington’s disease (An et al., 2014). The group used Cas9-mediated incorporation of polyQ repeats in the first exon of HTT gene. The group described recombination rates as high as 12% compared to less than 1% by other technologies. The first report on genetic correction of mutations in patient iPSC lines was reported by the Kan group (Xie et al., 2014). The group was looking at iPSC lines from patients with β-thalassemia, which is caused by mutations in the human hemoglobin beta (HBB) gene. iPSC lines were generated from the fibroblasts of a β-thalassemia patient doubly heterozygous for the −28 (A/G) mutation of the promoter and the 4-bp (TCTT) deletion at codons 41 and 42 of exon 2, both being common mutations in the Chinese population. They used three different gRNAs, which varied in efficiency in generating DSB from 5% to 23%, and the replacement gene was delivered using piggyBac transposons. They then differentiated these cell lines into the hematopoietic progenitors and erythroblasts to test the efficacy of gene correction. Upon analysis of HBB mRNA levels, they reported that the gene-corrected lines had 16-fold higher HBB levels compared to control lines. Similar results have been reported by others (Song et al., 2015). They also saw that gene-corrected lines had reduced reactive oxygen species production compared to control uncorrected iPSC lines. Another disease that has been corrected in iPSC-based in vitro models is severe combined immunodeficiency (SCID) (Chang et al., 2015). SCID is caused by mutations in the Janus family kinase member, JAK3. Correction of the JAK3 mutation by CRISPR–Cas9-enhanced gene targeting restored normal T cell development, including the production of mature T cell populations with a broad T cell receptor repertoire from the iPSCs following T cell-directed differentiation. The group also reported no off-target modification upon the whole genome sequencing. Others have modeled chronic granulomatous disease which is a rare genetic disease caused by the lack of an oxidative burst, normally performed by phagocytic cells (Flynn et al., 2015). The group used CRISPR-mediated homologous recombination to reintroduce a previously skipped exon in the cytochrome b-245 heavy chain protein. This resulted in the restoration of oxidative burst function in iPSC-derived phagocytes from the gene-corrected lines.
Analysis of coronavirus envelope protein with cellular automata model
Published in International Journal of Parallel, Emergent and Distributed Systems, 2022
Raju Hazari, Parimal Pal Chaudhuri
The hypothesis has been validated from mutational study on two proteins: (i) HBB beta-globin hemoglobin protein [3,36,37], (ii) mutations reported in COVID-2 infected patients [9,36]. For each of these case studies, difference of CAML model parameter between wild and mutant corroborates the results reported in vitro/in vivo studies in respect of deviation of structure–function of specific mutant from its wild leading to a specific disease.