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• W3182357811 abstract "The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna. They received the prestigious award for their groundbreaking description of the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) system as a genome editing tool in 2012.1 Since then, what was originally known as the adaptive antiviral immune system of bacteria, these “molecular scissors” have been exploited as a tool for genome editing by biomedical researchers working in fields as diverse as hematology, neuroscience and immunology, with several clinical trials planned or already ongoing for the treatment of diverse inborn pathologies2, 3 (also see NCT03872479, NCT04191148 and NCT04601051 at https://clinicaltrials.gov). CRISPR-mediated genome editing has been garnering interest in cancer therapy, particularly as a tool to generate “off-the-shelf” T-cell products that do not express major histocompatibility complex molecules, or to render T cells refractory to inhibitory signaling.4 The employment of T cells with transgenic T-cell receptors (TCRs) or chimeric antigen receptors (CARs) specific to tumor antigens in combination with CRISPR-mediated genome editing is particularly promising. This has resulted in stabilized transgene expression, enhanced T-cell effector function and/or increased efficacy in murine models of both blood cancers and solid tumors.4 Nonetheless, the generation of T cells with both knockouts and knock-ins is a laborious process. Furthermore, for targeting specific T-cell subsets, enrichment or cell sorting is required prior to genetic engineering. However, recent work published in Cell Reports by the aforementioned Nobel laureate Jennifer Doudna5 has started to pave the way for change. Electroporation is currently the dominant strategy for delivering Cas9 into cells. Usually, Cas9 is precoupled to the guide RNA and trans-activating CRISPR RNA that are used to target the Cas9 protein to and activate the Cas9 protein at the genomic locus of interest. The combined Cas9–RNA complex is referred to as a Cas9 ribonuclear protein (RNP), and Cas9 RNP electroporation was shown to be an effective method for the genetic modification of human T cells.6, 7 However, for large amounts of cells and/or targets, electroporation is impractical. Hamilton et al.5 report on a system that shuttles Streptococcus pyogenes Cas9 RNPs into cells utilizing retrovirus-like particles (VLPs; Figure 1a). To produce the Cas9 RNP-containing VLPs, the authors fused Cas9 to the Gag structural viral protein to ensure uptake into VLPs during viral assembly. While the use of VLPs as a vector to introduce Cas9 proteins into cells is not novel, the authors were able to produce VLPs that contain Cas9 RNPs. In contrast to previous reports using VLPs as a Cas9 carrier,8 this approach results in a VLP that contains both the Cas9 machinery and required RNAs (i.e. the guide RNA and trans-activating CRISPR RNA), and thus does not require the separate introduction of targeting guide RNAs. Furthermore, this new technique also allows for the simultaneous introduction of a transgene of interest, such as a tumor-specific TCR or CAR. Of note, by tagging the transgene via a self-cleaving peptide with a fluorescent marker, cells expressing the transgene can be easily enriched by, for example, fluorescent cell sorting. Lastly, what also makes this method unique is the ability to target specific cell subsets in a mixed population, at least in vitro. By making use of VLPs that have been pseudotyped with the envelope (Env) protein of the CD4-tropic HIV-1, they were able to produce VLPs that efficiently infect CD4+ T cells at a high specificity: approximately 50% of CD4+ T cells expressed a fluorescent reporter gene, compared with only about 2.5% of CD8+ T cells from the same well. Currently, T cells equipped with CARs are being employed for the treatment of several blood cancers.9 While this type of therapy is very efficient for blood cancers, CAR T cells are less effective for the treatment of solid tumors. Therefore, many groups have tried to enhance T-cell effector function by making use of CRISPR/Cas9-mediated genome editing in order to maximize T-cell killing potential. Ranging from direct augmentation of cytokine production by modulating TCR or costimulatory signaling to fine-tuning post-transcriptional regulation, or by simply knocking out inhibitory receptors such as PD-1,4 many of these approaches utilize a two-step process and required sequential genome editing and introduction of tumor antigen-specific CARs or TCRs. Instead, the novel method from the Doudna laboratory is able to do both simultaneously.5 Recently, the first-in-human clinical trial with CRISPR/Cas9-generated PD-1 knockout NY-ESO-1 TCR transgenic T cells was performed,10 where indeed subsequent electroporation and lentiviral transduction steps were required to introduce the PD-1 knockout and transgenic TCR, respectively. The VLP-mediated delivery of both the Cas9 machinery and transgene of interest could have significantly streamlined and optimized the production of the therapeutic T-cell product. Lastly, by targeting β2 microglobulin and/or T-cell receptor alpha chain (TRAC), as exemplified by Hamilton et al.,5 VLPs could also be used to produce “off-the-shelf” (CAR) T-cell products for adoptive therapy.4 Significantly, the approach of Hamilton et al. results in only transient presence of genome editing machinery components, limiting the risk for prolonged off-target effects as a result of sustained expression upon Cas9-transgene insertion. This is a huge benefit compared with lentiviral or retroviral delivery of Cas9 genes and guide RNAs, as the constitutive expression of genome editing machinery can result in off-target effects over time. While this can be mitigated with on/off switchable or cell-specific promoters, transient Cas9 presence is preferred. Furthermore, it has been shown that a majority of healthy individuals possess antibodies directed against Cas9 proteins,11 further highlighting the importance of transient Cas9 expression and/or presence, especially when employing the end product in therapeutic settings. Hamilton et al.12 exploited the cell tropism of HIV-1, but this can be expanded upon by pseudotyping VLPs with other viral envelope proteins, for example, the envelope protein of the rabies virus for targeting neural cells, or influenza for targeting airway epithelial cells. Nonetheless, the specific targeting of CD4+ T cells with CRISPR VLPs has broad applications (Figure 1b), ranging from the (in vivo) production of CAR T cells, where Cas9-mediated knockout of TRAC genes can stabilize transgene expression,4 to rendering CD4+ T cells of HIV-1-infected individuals refractive to infection by knocking out, for example, CCR5 and/or replacing it with the CCR5Δ mutant that does not allow cellular entry,13 to the correction of inborn errors affecting T-cell effector function14 by knocking out defective molecules and replacing them with functional copies. Of note, the HIV-1-pseudotyped version of the Cas9-VLP resulted in lower genome editing frequencies (approximately 14–28%) in primary human T cells compared with conventional methods such as electroporation.6, 7 This can potentially be increased by making use of recently described more efficient Cas9 molecules,15 but this remains to be determined. Furthermore, CD4-tropic HIV-1 strains have also been shown to infect macrophages, which should be taken into careful consideration, especially before implementing this tool for in vivo gene editing. Future research in humanized mice could shed light on in vivo safety and feasibility of Cas9-VLP-mediated genome editing. Nonetheless, the study by Hamilton et al. provides a novel angle for the therapeutic use of CRISPR-mediated genome editing and provides a solid base for future research. Author has no competing interests or disclosures to declare. Julian J Freen-van Heeren Conceptualization; Visualization; Writing-original draft; Writing-review & editing." @default.
• W3182357811 created "2021-07-19" @default.
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• W3182357811 date "2021-07-06" @default.
• W3182357811 modified "2023-10-17" @default.
• W3182357811 title "Exploiting HIV‐1 tropism to target CD4 + T cells for CRISPR" @default.
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• W3182357811 doi "https://doi.org/10.1111/imcb.12487" @default.
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