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29 pages, 6021 KB

Open AccessArticle

Identification and Characterization of Fully Human FOLR1-Targeting CAR T Cells for the Treatment of Ovarian Cancer

by Maria Bethke, Pierre Abramowski, Miriam Droste, André Felsberger, Lisa Kochsiek, Bettina Kotter, Luisa Plettig, Kateryna Antonova, Salpy Baghdo, Nico Burzan, Florian Tomszak, Manuel Martinez-Osuna, Dominik Eckardt and Christoph Herbel

Cells 2024, 13(22), 1880; https://doi.org/10.3390/cells13221880 - 14 Nov 2024

Cited by 2 | Viewed by 2925

Abstract

CAR T cell therapy has been an effective treatment option for hematological malignancies. However, the therapeutic potential of CAR T cells can be reduced by several constraints, partly due to immunogenicity and toxicities. The lack of established workflows enabling thorough evaluation of new candidates, limits comprehensive CAR assessment. To improve the selection of lead CAR candidates, we established a stringent, multistep workflow based on specificity assessments, employing multiple assays and technologies. Moreover, we characterized a human FOLR1-directed CAR binding domain. Selection of binding domains was based on extensive specificity assessment by flow cytometry and imaging, to determine on-/off-target and off-tumor reactivity. CAR T cell functionality and specificity were assessed by high-throughput screening and advanced in vitro assays. Our validation strategy highlights that assays comprehensively characterizing CAR functionality and binding specificity complement each other. Thereby, critical specificity considerations can be addressed early in the development process to overcome current limitations for future CAR T cell therapies. Full article

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15 pages, 3113 KB

Open AccessArticle

Off-Target-Based Design of Selective HIV-1 PROTEASE Inhibitors

by Gabriele La Monica, Antonino Lauria, Alessia Bono and Annamaria Martorana

Int. J. Mol. Sci. 2021, 22(11), 6070; https://doi.org/10.3390/ijms22116070 - 4 Jun 2021

Cited by 9 | Viewed by 4842

Abstract

The approval of the first HIV-1 protease inhibitors (HIV-1 PRIs) marked a fundamental step in the control of AIDS, and this class of agents still represents the mainstay therapy for this illness. Despite the undisputed benefits, the necessary lifelong treatment led to numerous severe side-effects (metabolic syndrome, hepatotoxicity, diabetes, etc.). The HIV-1 PRIs are capable of interacting with “secondary” targets (off-targets) characterized by different biological activities from that of HIV-1 protease. In this scenario, the in-silico techniques undoubtedly contributed to the design of new small molecules with well-fitting selectivity against the main target, analyzing possible undesirable interactions that are already in the early stages of the research process. The present work is focused on a new mixed-hierarchical, ligand-structure-based protocol, which is centered on an on/off-target approach, to identify the new selective inhibitors of HIV-1 PR. The use of the well-established, ligand-based tools available in the DRUDIT web platform, in combination with a conventional, structure-based molecular docking process, permitted to fast screen a large database of active molecules and to select a set of structure with optimal on/off-target profiles. Therefore, the method exposed herein, could represent a reliable help in the research of new selective targeted small molecules, permitting to design new agents without undesirable interactions. Full article

(This article belongs to the Special Issue Application of In Silico Techniques in Drug Design)

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21 pages, 2378 KB

Open AccessEditor’s ChoiceReview

Carbonic Anhydrase Inhibitors Targeting Metabolism and Tumor Microenvironment

by Andrea Angeli, Fabrizio Carta, Alessio Nocentini, Jean-Yves Winum, Raivis Zalubovskis, Atilla Akdemir, Valentina Onnis, Wagdy M. Eldehna, Clemente Capasso, Giuseppina De Simone, Simona Maria Monti, Simone Carradori, William A. Donald, Shoukat Dedhar and Claudiu T. Supuran

Metabolites 2020, 10(10), 412; https://doi.org/10.3390/metabo10100412 - 14 Oct 2020

Cited by 147 | Viewed by 8386

Abstract

The tumor microenvironment is crucial for the growth of cancer cells, triggering particular biochemical and physiological changes, which frequently influence the outcome of anticancer therapies. The biochemical rationale behind many of these phenomena resides in the activation of transcription factors such as hypoxia-inducible factor 1 and 2 (HIF-1/2). In turn, the HIF pathway activates a number of genes including those involved in glucose metabolism, angiogenesis, and pH regulation. Several carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA IX and XII, actively participate in these processes and were validated as antitumor/antimetastatic drug targets. Here, we review the field of CA inhibitors (CAIs), which selectively inhibit the cancer-associated CA isoforms. Particular focus was on the identification of lead compounds and various inhibitor classes, and the measurement of CA inhibitory on-/off-target effects. In addition, the preclinical data that resulted in the identification of SLC-0111, a sulfonamide in Phase Ib/II clinical trials for the treatment of hypoxic, advanced solid tumors, are detailed. Full article

(This article belongs to the Special Issue Metabolism in the Tumor Microenvironment)

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15 pages, 2064 KB

Open AccessArticle

Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing

by Wenjie Xu, Wei Fu, Pengyu Zhu, Zhihong Li, Chenguang Wang, Chaonan Wang, Yongjiang Zhang and Shuifang Zhu

Int. J. Mol. Sci. 2019, 20(17), 4125; https://doi.org/10.3390/ijms20174125 - 23 Aug 2019

Cited by 10 | Viewed by 4863

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been widely applied in functional genomics research and plant breeding. In contrast to the off-target studies of mammalian cells, there is little evidence for the common occurrence of off-target sites in plants and a great need exists for accurate detection of editing sites. Here, we summarized the precision of CRISPR/Cas9-mediated mutations for 281 targets and found that there is a preference for single nucleotide deletions/insertions and longer deletions starting from 40 nt upstream or ending at 30 nt downstream of the cleavage site, which suggested the candidate sequences for editing sites detection by whole-genome sequencing (WGS). We analyzed the on-/off-target sites of 6 CRISPR/Cas9-mediated Arabidopsis plants by the optimized method. The results showed that the on-target editing frequency ranged from 38.1% to 100%, and one off target at a frequency of 9.8%–97.3% cannot be prevented by increasing the specificity or reducing the expression level of the Cas9 enzyme. These results indicated that designing guide RNA with high specificity may be the preferred factor to avoid the off-target events, and it is necessary to predict or detect off-target sites by WGS-based methods for preventing off targets caused by genome differences in different individuals. Full article

(This article belongs to the Section Molecular Plant Sciences)

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18 pages, 1604 KB

Open AccessArticle

Modulation of the RNA Interference Activity Using Central Mismatched siRNAs and Acyclic Threoninol Nucleic Acids (aTNA) Units

by Adele Alagia, Montserrat Terrazas and Ramon Eritja

Molecules 2015, 20(5), 7602-7619; https://doi.org/10.3390/molecules20057602 - 24 Apr 2015

Cited by 16 | Viewed by 8715

Abstract

The understanding of the mechanisms behind nucleotide recognition by Argonaute 2, core protein of the RNA-induced silencing complex, is a key aspect in the optimization of small interfering RNAs (siRNAs) activity. To date, great efforts have been focused on the modification of certain regions of siRNA, such as the 3'/5'-termini and the seed region. Only a few reports have described the roles of central positions flanking the cleavage site during the silence process. In this study, we investigate the potential correlations between the thermodynamic and silencing properties of siRNA molecules carrying, at internal positions, an acyclic L-threoninol nucleic acid (aTNA) modification. Depending on position, the silencing is weakened or impaired. Furthermore, we evaluate the contribution of mismatches facing either a natural nucleotide or an aTNA modification to the siRNA potency. The position 11 of the antisense strand is more permissive to mismatches and aTNA modification, in respect to the position 10. Additionally, comparing the ON-/OFF-target silencing of central mismatched siRNAs with 5'-terminal modified siRNA, we concluded: (i) central perturbation of duplex pairing features weights more on potency rather than silencing asymmetry; (ii) complete bias for the ON-target silencing can be achieved with single L-threoninol modification near the 5'-end of the sense strand. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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