The use of the powerful and programmable CRISPR-Cas system has greatly improved gene therapy, enabling accurate cancer modeling and providing a variety of genetic manipulations for cancer treatment and mutation detection. Since the RNA programmable gene editing of the Type II CRISPR-Cas9 system in SpyCas9, many CRISPR-Cas tools have been rapidly developed for gene therapy. For example, the CRISPR therapeutics method is mainly divided into four areas: (i) ex vivo approaches involving gene editing of hematopoietic cells for hemoglobinopathies, (ii) ex vivo methods in immuno-oncology for cancer, (iii) in vivo methods targeting liver (haemophilia) and (iv) additional in vivo approaches targeting other organ systems, such as muscle (DMD) and lung (CF).
Although the conventional nuclease-based gene targeting technologies, TALENs and ZFNs, have been explored for therapeutic gene editing previously, CRISPR-based cancer therapeutics already represent the majority (79%) of the gene editing cancer trials worldwide. Recent advances and success in the ﬁeld of cancer immunotherapy highlight the therapeutic potential of engineering chimeric antigen receptor (CAR) T cells. To further enhance the efﬁcacy and safety of T cell therapeutics, gene editing technologies, including zinc ﬁnger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9, have been recently used to genetically modify primary human T cells.
Compared with ZFNs and TALENs, CRISPR-Cas9 system provides more rapid and efﬁcient genetic manipulations, with multiplexed editing capability, simply by viral gene delivery systems or physical transfection methods such as electroporation of Cas9 and sgRNA expression constructs, or Cas9 ribonucleoproteins (RNPs). This advantage allows rapid T cell manufacturing to boost T cell efﬁcacy by eliminating the genes such as programmed cell death protein 1 (PD-1) or cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which encode T cell inhibitory receptors or signaling molecules.
Fig 1. Applications of CRISPR-Cas in Cancer Therapeutics.
Viral delivery systems for CRISPR-Cas9 include lentivirus, adeno-virus, and adeno-associatedvirus (AAV). Among these, AAVs are currently the most advanced methodology for in vivo gene delivery, and the efﬁcacy and safety of which have been testedin clinical trials and has been approved recently. In contrast to AAVs, nanoparticle-based delivery of CRISPR-Cas components has high loading capacity for nucleic acid cargos, without the risk of genomic integration and effects from persistent expression of CRISPR-Cas9. Cas9-sgRNA RNP complexes can be efﬁciently delivered by cationic lipids into the mouseinner ear cells in vivo to ameliorate hearing loss. In addition to nanoparticles, Cas9 RNP complexes can be directly delivered across the cellmembrane by co-delivery with small peptides or by modifying the electrostatic charge of theprotein.
Together, these therapeutic approaches using CRISPR-Cas9, including ex vivo and in vivogenome editing, wil greatly facilitate the rapid genetic manipulation of immune or cancer cells. Future efforts to build CRISPR-based gene perturbation circuits into CAR-T cells for increased programmability can also lead to more efﬁcient and safe CRISPR therapies and, consequently, to programmable and smart cellular medicines for cancer treatment.
In addition to expanding the CRISPR toolbox with new molecular gadgets, identifying and minimizing off-target effects induced by CRISPR-Cas systems are important to accelerate the therapeutic applications. Using in silico prediction of potential off-target cleavage sites, followed by experimental validation of nonhomologous end joining (NHEJ)-induced indel mutations, these mismatched sites are found to be mutagenized at similar frequencies as the intended ones. Based on these ﬁndings and the molecular features of single guide RNA (sgRNA), a number of online tools, such as the CRISPR Design Tool, E-CRISP, CRISPRscan, GuideScan, Cas-OFFinder, and CRISPR ML, were designed to take into account the sequence similarity and other parameters for the prediction of potential off-target sites.
While, a comprehensive examination of off-targeted sites is still indispensable. Also, the effects of human genetic variation on creating and destroying PAMs and on- or off-target sites also need to be taken into account to minimizethe risk of adverse outcomes and treatment failure from CRISPR-based therapy. Anti-CRISPR proteins as off-switches for CRISPR-Cas activity. This is particularly important for therapeutic applications where anti-CRISPR proteins can be harnessed to not only reduce off-target editing but also to disable Cas9 activity. Although further optimization is required to apply anti-CRISPR systemsfor clinical use, they provide promise of the addition of another security checkpoint for CRISPR therapeutics.
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