Cas9‐Cleavage Sequences in Size‐Reduced Plasmids Enhance Nonviral Genome Targeting of CARs in Primary Human T Cells

Jing, Ruirui; Jiao, Peng; Chen, Jiangqing; Meng, Xianhui; Wu, Xiaoyun; Duan, Yanran; Shang, Kai; Qian, Liling; Huang, Yanjie; Liu, Junwei; Huang, Tao; Jin, Jin; Chen, Wei; Zeng, Xun; Yin, Weiwei; Gao, Xiaofei; Zhou, Chun; Sadelain, Michel; Sun, Jie

doi:10.1002/smtd.202100071

Cited by 24 publications

(17 citation statements)

References 27 publications

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“…As a result of different optimization steps, in most publications on virus-free TCR/CAR knock-ins blunt-ended dsDNA or plasmids were used with the frequency of knock-in T cells reported in a range between 5-50% after 7-14 days of expansion (49,50,51,52,53). Based on experience of authors of this review, the number of recovered T cells 10 days after initiation of the editing procedure can reach 10-200 times the number of PBMCs used as input (51).…”

Section: Transgene "Knock-in" With Crispr-cas Gene Editingmentioning

confidence: 99%

Review: Sustainable Clinical Development of CAR-T Cells – Switching From Viral Transduction Towards CRISPR-Cas Gene Editing

et al. 2022

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T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first gene-modified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

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Section: Transgene "Knock-in" With Crispr-cas Gene Editingmentioning

confidence: 99%

Review: Sustainable Clinical Development of CAR-T Cells – Switching From Viral Transduction Towards CRISPR-Cas Gene Editing

et al. 2022

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“…Modified guide RNAs (gRNAs) was synthesized by GeneScript. Guide RNAs were reconstituted at 1μg/μL in RNase free water, Cas9 proteins were produced by our lab as described in our previous work [32] , In brief, they were complexed in 2:1 gRNA to Cas9 molar ratio at room temperature for 20 min then electroporated into T cells immediately after complexing [32] .…”

Section: Methodsmentioning

confidence: 99%

“…Based on pAAV-TRAC-1928z plasmid (as described in our previous work [32] , named it as CD19-WT) we designed and cloned the pAAV-TRAC-1928z-1XX (named as CD19-1XX). Briefly, the CD19 CAR comprises a single chain variable fragment [19] scFv specific for the human CD19 (AXL CAR comprises a single chain variable fragment of anti-AXL [33] specific binding to human AXL), preceded by a CD8a leader peptide and followed by CD28 hinge, transmembrane and intracellular regions and CD3ζ intracellular domain.…”

Section: Methodsmentioning

confidence: 99%

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Balancing activation and costimulation of CAR tunes signaling dynamics and enhances therapeutic potency

Sun

Duan

Chen

et al. 2022

Preprint

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Background: Primary human T cells engineered with chimeric antigen receptors (CARs) ex vivo can be adoptively transferred to treat cancer. CD19-targeting CAR with CD28 costimulatory domain and CD3ζ activation domain have been approved by the US FDA for treating B cell malignancies. Methods: Here we generated mutation of immunorecpetor tyrosine-based activation motifs (ITAMs) in CD3ζ, namely 1XX CAR, which altered the balance of activation and costimulation. Next we investigated whether 1XX design could enhance therapeutic potency against solid tumors. We constructed both CD19- and AXL-specific 1XX CARs and compared their in vitro and in vivo functions with their WT counterparts. Results: Even though 1XX CARs decreased cytotoxicity against tumor cells in vitro, they showed better anti-tumor efficacy in both pancreatic and melanoma mouse models. Detailed analysis revealed that 1XX CAR-T cells proliferated more in response to antigen stimulation in vitro, persisted longer in vivo and had higher percentage of central memory cells. As 1XX modification directly calibrates CAR activation potential, we utilized fluorescence resonance energy transfer (FRET)-based biosensor to monitor signaling dynamics downstream of CARs. Decreased ITAM numbers in 1XX resulted in similar ZAP70 activation, while 1XX induced higher Ca2+ elevation and faster Erk activation than WT CAR, which may contribute to the better therapeutic potency of 1XX. Conclusions: Our results established the surpiosity of 1XX against two targets in different solid tumor models and shed light on the underlying molecular mechanism of CAR signaling, paving the way for the clinical application of 1XX CARs against solid tumors.

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“…An increase in insertions and deletions without a decrease in level of activity demonstrates how these modifications improve the nuclease’s editing efficiency. Jing et al (2021) included the same MS modification to their sgRNA and observed a 15% higher knock-in efficiency compared to unmodified sgRNA when attempting to knock-in predetermined template plasmids into primary human T cells. Ryan et al (2022) similarly engineered the MS and MP nucleotide modifications to their synthesized sgRNA structures and observed increased indel editing for the HBB gene within human primary T cells.…”

Section: Gene Editing Cargomentioning

confidence: 99%

Delivering the CRISPR/Cas9 system for engineering gene therapies: Recent cargo and delivery approaches for clinical translation

Foley

Sims

Duggan

et al. 2022

Front. Bioeng. Biotechnol.

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Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9) has transformed our ability to edit the human genome selectively. This technology has quickly become the most standardized and reproducible gene editing tool available. Catalyzing rapid advances in biomedical research and genetic engineering, the CRISPR/Cas9 system offers great potential to provide diagnostic and therapeutic options for the prevention and treatment of currently incurable single-gene and more complex human diseases. However, significant barriers to the clinical application of CRISPR/Cas9 remain. While in vitro, ex vivo, and in vivo gene editing has been demonstrated extensively in a laboratory setting, the translation to clinical studies is currently limited by shortfalls in the precision, scalability, and efficiency of delivering CRISPR/Cas9-associated reagents to their intended therapeutic targets. To overcome these challenges, recent advancements manipulate both the delivery cargo and vehicles used to transport CRISPR/Cas9 reagents. With the choice of cargo informing the delivery vehicle, both must be optimized for precision and efficiency. This review aims to summarize current bioengineering approaches to applying CRISPR/Cas9 gene editing tools towards the development of emerging cellular therapeutics, focusing on its two main engineerable components: the delivery vehicle and the gene editing cargo it carries. The contemporary barriers to biomedical applications are discussed within the context of key considerations to be made in the optimization of CRISPR/Cas9 for widespread clinical translation.

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Cas9‐Cleavage Sequences in Size‐Reduced Plasmids Enhance Nonviral Genome Targeting of CARs in Primary Human T Cells

Cited by 24 publications

References 27 publications

Review: Sustainable Clinical Development of CAR-T Cells – Switching From Viral Transduction Towards CRISPR-Cas Gene Editing

Review: Sustainable Clinical Development of CAR-T Cells – Switching From Viral Transduction Towards CRISPR-Cas Gene Editing

Balancing activation and costimulation of CAR tunes signaling dynamics and enhances therapeutic potency

Delivering the CRISPR/Cas9 system for engineering gene therapies: Recent cargo and delivery approaches for clinical translation

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