Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

Hale, Malika; Lee, Baeckseung; Honaker, Yuchi; Leung, Wing‐Hung; Grier, Alexandra E.; Jacobs, Holly M.; Sommer, Karen; Sahni, Jaya; Jackson, Shaun W.; Scharenberg, Andrew M.; Astrakhan, Alexander

doi:10.1016/j.omtm.2016.12.008

Cited by 57 publications

(48 citation statements)

References 41 publications

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“…The CAR T-cell manufacturer was alerted to the HIV status of the patients and agreed to manufacture commercial product to treat their refractory B-cell lymphomas. 9 Given the number of outstanding questions regarding CAR T-cell therapy in HIV-positive patients with hematologic malignancies, these patients ideally would be studied within the context of prospective clinical trials. Although patients who are positive for HIV are not expressly excluded from the US Food and Drug Administration label for the approved CAR T-cell products in lymphoma, to the best of our knowledge they have been excluded from the pivotal clinical trials that established their safety and efficacy.…”

Section: Discussionmentioning

confidence: 99%

“…For example, gene editing technology has been used to direct the insertion of the anti-CD19 CAR gene into the CCR5 locus, rendering that T cell resistant to entry of the HIV virus, as well as targeted against B-cell lymphoma. 9 Given the number of outstanding questions regarding CAR T-cell therapy in HIV-positive patients with hematologic malignancies, these patients ideally would be studied within the context of prospective clinical trials. This also may help to address disparities in the management of HIV-infected patients with cancer.…”

Section: Discussionmentioning

confidence: 99%

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Successful anti‐CD19 CAR T‐cell therapy in HIV‐infected patients with refractory high‐grade B‐cell lymphoma

Abramson

Irwin

Frigault

et al. 2019

Cancer

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Successful anti‐CD19 CAR T‐cell therapy in HIV‐infected patients with refractory high‐grade B‐cell lymphoma

Abramson

Irwin

Frigault

et al. 2019

Cancer

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“…These cells were shown to be more potent than conventional lentivirally transduced CART cells because of a more physiological – TCR-like regulation of CAR expression. [58, 59]…”

Section: Strategies To Generate Universal Cartmentioning

confidence: 99%

Next-Generation Chimeric Antigen Receptor T-Cell Therapy: Going off the Shelf

2017

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Autologous, patient-specific chimeric antigen receptor T cell (CART) therapy has emerged as a powerful and potentially curative therapy for cancer, especially for CD19-positive hematological malignancies. Indeed, CD19-directed CART (CART-19) cell therapy (tisagenlecleucel-t) was Food and Drug Administration (FDA) approved for acute lymphoblastic leukemia on August 30, 2017 and approval of CART-19 in B-cell lymphomas is expected in late 2017. The development of this technology and its wider application is partly limited by the patient-specificity nature of such a platform and by the time required for CART manufacturing. The efficacious generation of universal allogeneic CART cells would overcome these limitations and represent a major advance in the field. However, several obstacles in the generation of universal CART cells need to be overcome, namely the risk of rejection of CART by the recipient and the risk of graft versus host disease mediated by the allogeneic CART. In this review, we discuss the different strategies being employed to generate universal CART and discuss our perspective on the successful development of a truly off-the-shelf CART product.

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“…In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homologydirected repair (HDR), but to date in human T cells this still requires viral transduction 8,9 . Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function.…”

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confidence: 99%

Reprogramming human T cell function and specificity with non-viral genome targeting

Roth

Puig-Saus

et al. 2017

Preprint

150

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The major barrier to effective non-viral T cell genome targeting of large DNA sequences has been the toxicity of the DNA 10 . While the introduction of short singlestranded oligodeoxynucleotide (ssODN) HDR templates does not cause significant T cell death, it has been shown that larger linear double stranded (dsDNA) templates are toxic at high concentrations 11,12 . Contrary to expectations, we found that co-electroporation of human primary T cells with CRISPR-Cas9 ribonucleoprotein (Cas9 RNP 13,14 ) complexes and long (>1kb) linear dsDNA templates reduced the toxicity associated with the dsDNA template (Extended Data Fig 1). Cas9 RNPs were co-electroporated with a dsDNA HDR template designed to introduce an N-terminal GFP-fusion in the housekeeping peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/183418 doi: bioRxiv preprint first posted online Aug. 31, 2017; 3 gene RAB11A (Fig. 1a). Systematic exploration of this approach while optimizing for both viability and efficiency ( Fig. 1b and Extended Data Fig. 2) resulted in GFP expression in ~50% of cells in both primary human CD4+ and CD8+ T cells. The method was reproducibly efficient while maintaining high cell viability and expandability (Fig. 1c, d, e, and Extended Data Fig. 3). The system is also compatible with current manufacturing protocols for cell therapies as it could be applied to fresh or cryopreserved cells, bulk T cells or FACS-sorted sub-populations, and cells from whole blood or leukapheresis (Extended Data Fig. 4).We next confirmed that the system could be applied broadly by targeting sequences in different locations throughout the genome. We efficiently engineered GFP+ primary T cells by generating fusions with different genes (Fig. 2a and Fig. 3a and Extended Data Fig. 14). One mutation, c.530A>G, creates a premature stop codon. With non-viral genome targeting, we were able to correct the mutation and observe IL2RA expression on the surface of corrected T cells from the patient (Fig. 3b). Long dsDNA templates led to efficient correction of the mutations. Because only two base pair changes were necessary (one to correct the mutation and one to silently remove the gRNA's PAM sequence), a short single-stranded DNA (~120 bps) could also be used to make the correction. These single-stranded DNAs were able to correct the mutation at high frequencies, although the efficiency of correction was lower than with the longer dsDNA template (Extended Data Fig. 15, 16).Correction was successful in T cells from all three siblings, but lower rates of IL2RA expression were seen in compound het 3, which could be due to altered cell-state associated with the patient's disease or the fact she was the only sibling treated with immunosuppressive therapy (Extended Data Table 1 and Extended Data Fig. 17). The second mutation identified, c.800delA, causes a frameshift in the reading frame of the final IL2RA exon. This frameshift mutation c...

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Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

Cited by 57 publications

References 41 publications

Successful anti‐CD19 CAR T‐cell therapy in HIV‐infected patients with refractory high‐grade B‐cell lymphoma

Successful anti‐CD19 CAR T‐cell therapy in HIV‐infected patients with refractory high‐grade B‐cell lymphoma

Next-Generation Chimeric Antigen Receptor T-Cell Therapy: Going off the Shelf

Reprogramming human T cell function and specificity with non-viral genome targeting

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