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      Viral vectors and extracellular vesicles: innate delivery systems utilized in CRISPR/Cas-mediated cancer therapy

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          Abstract

          Gene editing-based therapeutic strategies grant the power to override cell machinery and alter faulty genes contributing to disease development like cancer. Nowadays, the principal tool for gene editing is the clustered regularly interspaced short palindromic repeats-associated nuclease 9 (CRISPR/Cas9) system. In order to bring this gene-editing system from the bench to the bedside, a significant hurdle remains, and that is the delivery of CRISPR/Cas to various target cells in vivo and in vitro. The CRISPR-Cas system can be delivered into mammalian cells using various strategies; among all, we have reviewed recent research around two natural gene delivery systems that have been proven to be compatible with human cells. Herein, we have discussed the advantages and limitations of viral vectors, and extracellular vesicles (EVs) in delivering the CRISPR/Cas system for cancer therapy purposes.

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Martin Jinek, Krzysztof Chylinski, Ines Fonfara … (2012)
          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
          Article 0 comments Cited 3149 times – based on 0 reviews     Review now
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            Multiplex genome engineering using CRISPR/Cas systems.

            Le Cong, F. Ran, David T. Cox … (2013)
            Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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              Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.

              Hadi Valadi, Karin Ekström, Apostolos Bossios … (2007)
              Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).
              Article 0 comments Cited 2426 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Majid Safa:
                ORCID: http://orcid.org/0000-0003-0070-6620
                majidsafa@gmail.com
                Journal
                Journal ID (nlm-ta): Cancer Gene Ther
                Journal ID (iso-abbrev): Cancer Gene Ther
                Title: Cancer Gene Therapy
                Publisher: Nature Publishing Group US (New York )
                ISSN (Print): 0929-1903
                ISSN (Electronic): 1476-5500
                Publication date (Electronic): 28 February 2023
                Pages: 1-19
                Affiliations
                [1 ]GRID grid.411746.1, ISNI 0000 0004 4911 7066, Department of Hematology and Blood Banking, Faculty of Allied Medicine, , Iran University of Medical Sciences, ; Tehran, Iran
                [2 ]GRID grid.411230.5, ISNI 0000 0000 9296 6873, School of Medicine, , Ahvaz Jundishapur University of Medical Sciences, ; Ahvaz, Iran
                [3 ]Division of Clinical Laboratory, Tehran Hospital of Petroleum Industry, Tehran, Iran
                [4 ]GRID grid.411600.2, Department of Immunology, School of Medicine, , Shahid Beheshti University of Medical Sciences, ; Tehran, Iran
                [5 ]GRID grid.411600.2, Department of Clinical Biochemistry, School of Medicine, , Shahid Beheshti University of Medical Sciences, ; Tehran, Iran
                Author information
                http://orcid.org/0000-0002-1227-491X
                http://orcid.org/0000-0003-0070-6620
                Article
                Publisher ID: 597
                DOI: 10.1038/s41417-023-00597-z
                PMC ID: 9971689
                PubMed ID: 36854897
                SO-VID: 8b2cfd76-d3bb-4d24-b482-858c323f3efa
                Copyright © © The Author(s), under exclusive licence to Springer Nature America, Inc. 2023, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
                License:

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                Date received : 9 October 2022
                Date revision received : 13 January 2023
                Date accepted : 1 February 2023
                Categories
                Subject: Review Article

                ScienceOpen disciplines: Oncology & Radiotherapy
                Keywords: genetic vectors,cancer therapy
                Data availability:
                ScienceOpen disciplines: Oncology & Radiotherapy
                Keywords: genetic vectors, cancer therapy

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