A highly efficient transgene knock-in technology in clinically relevant cell types

Allen, Alex; Khan, Samia Q.; Margulies, Carrie M; Viswanathan, Ramya; Lele, Swarali Surendra; Blaha, Laura; Scott, Sean N.; Izzo, Kaitlyn; Gerew, Alexandra; Pattali, Rithu; Cochran, Nadire; Holland, Carl S; Zhao, Amy H.; Sherman, Stephen E.; Jaskolka, Michael C; Wu, Meng; Wilson, Aaron; Sun, Xiaoqi; Ciulla, Dawn; Zhang, Deric; Nelson, Jacqueline D; Zhang, Peisheng; Mazzucato, Patrizia; Huang, Yan; Giannoukos, Georgia; Marco, Eugenio; Nehil, Michael; Follit, John A; Chang, Kai‐Hsin; Shearman, Mark S.; Wilson, Christopher J.; Zuris, John A.

doi:10.1038/s41587-023-01779-8

Cited by 23 publications

(21 citation statements)

References 62 publications

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“…Surface tags and drug resistance cassettes have been used to enrich for cells with transgene integrations, but subjecting cells to multiple drugs or performing sequential rounds of positive selection can negatively impact cell viability, performance, and yield [16][17][18][19] . Alternatively, the targeting of essential loci has been used to enrich for cells with transgene integrations 20 , but the consequences of simultaneously editing multiple essential genes have not yet been evaluated. As editing outcomes at distinct loci are linked, previous studies have used a selective marker introduced at one locus to enrich for integrations at another locus 15,21,22 .…”

Section: Mainmentioning

confidence: 99%

Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection

Chang,

Vykunta,

Goodman

et al. 2024

Preprint

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Multiplexed reprogramming of T cell specificity and function can generate powerful next-generation cellular therapies. However, current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here, we develop a one-step process to enrich for unlabeled cells with knock-ins at multiple target loci using a family of repair templates namedSyntheticExon/Expression Disruptors (SEEDs). SEED engineering associates transgene integration with the disruption of a paired endogenous surface protein, allowing non-modified and partially edited cells to be immunomagnetically depleted (SEED-Selection). We design SEEDs to fully reprogram three critical loci encoding T cell specificity, co-receptor expression, and MHC expression, with up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). These methods are simple, compatible with existing clinical manufacturing workflows, and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.

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Section: Mainmentioning

confidence: 99%

Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection

Chang,

Vykunta,

Goodman

et al. 2024

Preprint

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show abstract

“…Safe harbor loci may also allow expression of CAR/TruC in Tregs without affecting TCR-expression, but these sites would require integration of larger transgene expression cassettes and do not enable automatic selection 47 . Essential genes, such as GAPDH , may be adopted for automatic selection, but fail to provide natural regulation of CAR expression 48 . Future studies may be required to evaluate whether the natural regulation of CD3ε -TruC provides an advantage when compared to stable overexpression.…”

Section: Discussionmentioning

confidence: 99%

Gene editing ofCD3 epsilongene to redirect regulatory T cells for adoptive T cell transfer

Du,

Noyan,

McCallion

et al. 2024

Preprint

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Adoptive transfer of regulatory T cells (Tregs) is a promising strategy to combat immunopathologies in transplantation and autoimmune diseases. Antigen-specific Tregs are more effective in modulating undesired immune reactions, but their low frequency in peripheral blood poses challenges for manufacturing and their clinical application. Chimeric antigen receptors (CARs) to redirect T-cell specificity have been applied to Tregs using retroviral vectors. However, retroviral gene transfer is costly, time consuming, and raises safety issues. Here, we explored non-viral gene editing to redirect Tregs with CARs, using HLA-A2-specific constructs for proof-of-concept studies in transplantation models. We introduce a virus-free CRISPR-Cas12a approach to integrate an antigen-binding domain into theCD3 epsilon(CD3ϵ) gene, generating Tregs expressing a T cell receptor fusion construct (TruC). TheseCD3ϵ-TruC Tregs exhibit potent antigen-dependent activation while maintaining responsiveness to TCR/CD3 stimulation. This enables preferential enrichment of TruC-redirected Tregs via repetitive CD3/CD28-stimulation in a GMP-compatible expansion system. Non-viral gene editedCD3ϵ-TruC Tregs retained their canonical phenotypic, epigenetic, and functional identity. In a humanized mouse model, HLA-A2-specificCD3ϵ-TruC Tregs demonstrate superior protection of allogeneic HLA-A2+ skin grafts from rejection compared to polyclonal Tregs. This approach provides a pathway for developing clinical-gradeCD3ϵ-TruC-based Tregs cell products for transplantation immunotherapy and other immunopathologies.

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“…35 Though NK cells have previously been genetically modified to successfully improve cytotoxicity and persistence using viral vectors, transposons, mRNA transfection, and CRISPR+AAV technologies, these methods elicit concerns of safety, lack the machinery for stable editing, or have knock-in limitations on transgene size. 19,20,[36][37][38][39] Here, we demonstrate an optimized CRISPR-Cas9-enabled approach that generates stable and precisely edited primary NK cells without the use of viral vectors. Our data show that the CRISPR platform can be leveraged for efficient knock-out of the inhibitory NKG2A receptor and virus-free knockin of an anti-GD2 CAR.…”

Section: Discussionmentioning

confidence: 99%

Virus-free CRISPR knock-in of a chimeric antigen receptor intoKLRC1generates potent GD2-specific natural killer cells

Shankar,

Zingler-Hoslet,

Shi

et al. 2024

Preprint

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Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR/Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knock-in of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-Seq, in-out PCR, and next generation sequencing. KLRC1-GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2+ human melanoma cells. Notably, KLRC1-GD2 CAR NK cells overcome HLA-E-based inhibition by HLA-E-expressing, GD2+ melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR/Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E+ solid tumors.

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A highly efficient transgene knock-in technology in clinically relevant cell types

Cited by 23 publications

References 62 publications

Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection

Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection

Gene editing ofCD3 epsilongene to redirect regulatory T cells for adoptive T cell transfer

Virus-free CRISPR knock-in of a chimeric antigen receptor intoKLRC1generates potent GD2-specific natural killer cells

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