For the generation for CAR and TCR T-cell therapies, RNP electrotransfer can simply be applied about 1 day prior to standard viral transduction of CAR and TCR transgenes.ĬRISPR can be used to engineer T cells to overcome key pathways of immunosuppression that lead to T cell–intrinsic resistance of cancer to ACTs. Importantly, applying this technique is feasible within the constraints and timelines of clinical-grade cell therapy manufacturing in compliance with Good Manufacturing Practices, and incorporating a CRISPR gene-editing step is not expected to increase overall production time ( 33). Cas9 delivered via transfection is degraded within 48 to 72 hours, which limits off-target editing and concerns about downstream Cas9 immunogenicity ( 32). Electrotransfer of RNP complexes into primary T cells can be accomplished efficiently without sacrificing cell viability, resulting in highly efficient gene knockout that is observable within about 48 hours. These challenges were overcome with the development of methods to transfect primary T cells with Cas9 ribonucleoprotein (RNP) complexes comprising recombinant Cas9 protein bound to a gRNA ( 28). For an in-depth comparison of CRISPR, ZFNs, and TALENs, readers are referred to a review by Khan ( 25). Recent technological advances have substantially reduced the risk of off-target mutagenesis with CRISPR, as discussed later in this review. Specificity is a key safety consideration for clinical applications as off-target mutagenesis poses a risk of oncogenic transformation in genetically engineered cells. Finally, the efficiency and specificity of genome editing via CRISPR are generally superior to those of ZFNs and TALENs ( 25, 26). This enables (i) rapid design and implementation of new CRISPR gene knockout/knock-in strategies, (ii) efficient multiplexing via incorporation of multiple gRNAs, and (iii) application of whole-genome CRISPR libraries for high-throughput screening. In contrast, the CRISPR system can be designed to target virtually any genomic sequence simply by altering the gRNA sequence. ZFNs and TALENs have limited potential for multiplexed genome editing and are not amenable to the generation of large-scale libraries ( 25). As a result, new ZFN or TALEN proteins must be engineered for each new target site, and design restrictions (particularly for ZFNs) limit the number of sites that can be targeted efficiently ( 24). ZFNs and TALENs rely on protein–DNA binding to guide site-specific DNA cleavage. CRISPR: Why and HowĬRISPR offers a number of notable advantages over earlier genome-editing technologies, such as zinc finger nucleases (ZFN) and transcription activator–like effector nucleases (TALEN). ![]() Emerging applications of CRISPR in non-T cell–based ACTs are also described. The purpose of this review is to detail recent applications of CRISPR that have advanced the field of cellular therapy, beginning with a discussion of specific genetic manipulations that hold the potential to enhance T-cell potency, safety, and scalability and ending with a review of CRISPR screening efforts that have unveiled novel targets for cell therapy enhancement. ![]() ![]() CRISPR genome-editing techniques are at the forefront of this revolution, offering a versatile platform for applications ranging from multiplexed gene knockout to site-specific transgene insertion to genome-wide genetic screening. Furthermore, genetic engineering approaches that enable use of allogeneic cell sources hold the potential to substantially improve the scalability of cell therapy manufacturing. This has initiated the next generation of cellular products that harbor creative genetic manipulations to improve T cells' potency and safety profiles and that can mitigate immunosuppressive triggers in the tumor microenvironment. As genome-editing technologies have become increasingly accessible, their use in cellular therapy has expanded greatly.
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