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Abstract
Therapeutic genome editing technology has been widely used as a powerful tool for directly correcting genetic mutations in target pathological tissues and cells to cure of diseases. The modification of specific genomic sequences can be achieved by utilizing programmable nucleases, such as Meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeat-associated nuclease Cas9 (CRISPR/Cas9). However, given the properties, such as large size, negative charge, low membrane penetrating ability, as well as weak tolerance for serum, and low endosomal escape, of these nucleases genome editing cannot be successfully applied unless in vivo delivery of related programmable nucleases into target organisms or cells is achieved. Here, we look back at delivery strategies having been used in the in vivo delivery of three main genome editing nucleases, followed by methodologies currently undergoing testing in clinical trials, and potential delivery strategies provided by analyzing characteristics of nucleases and commonly used vectors.
Highlights
Therapeutic genome editing is mediated by sequence-specific targeting nucleases, known as programmable nucleases
Much research has shown that zinc finger nucleases (ZFNs) can mediate genome editing efficiently by making site-specific DSBs inducing homology-directed repair (HDR) in target cells and the appropriate alternative and development of the in vivo delivery system is critical to clean barriers hampered on its clinical translation way
To deliver genome editing nucleases effectively, lentivirus have been developed for several generations from the early generation which contains the gene of cis-acting elements, such as the long terminal repeats to the second generation in which multiple non-essential accessory genes are deleted and self-inactivating vectors are established in order to avoid the unwanted generation of replication-competent lentiviral vectors (LVs) and the activation of nearby genes due to genomic integration and the lentiviral trans-activator of transcription-independent vectors were generated in the third generation
Summary
Therapeutic genome editing is mediated by sequence-specific targeting nucleases, known as programmable nucleases. Clinical applications of these programmable nuclease complexes are hampered by their inability to reach the intended target tissue, cross the cell membrane, and exert their therapeutic activities in vivo Both physical methods and delivery vectors are employed in the delivery of nuclease-based genome editing system (Figure 2). Being exploited as a “Trojan Horse” for genome therapeutic technologies, viral vectors whose parental wild-type viruses are rearranged to hinder replication or generation of infectious virions On the contrary, their ability of delivery nucleic acids for reaching and penetrating specific target cells and expressing genetic information in these cells is maintained [10]. As the delivery vector of ZFNs technology, lentiviral vectors which can accommodate sequences up to around 10 kilobases (kb) theoretically allow for site-specific genome modification or addition in predefined genomic sites They have been avoided because of multiple tragedies involving patient death in earlier clinical trials [36,37]. There is one study on the in vivo modification of a humanized mouse model of hemophilia B: in this study, the introduction of AAV on ZFNs achieved up to 45% site-specific cleavage of the hepatocytes [46]
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