Integration of ζ-deficient CARs into theCD3-zetagene conveys potent cytotoxicity in T and NK cells

Abstract

I.AbstractChimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the humanCD3ζ(CD247)gene, functional CAR fusion-genes are generated that exploit the endogenousCD3ζ gene as the CAR’s activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells,CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings.CD3ζ-CD19-CAR-T cells exhibited comparable leukemia control toT cell receptor alpha constant(TRAC)-replaced and lentivirus-transduced CAR-T cellsin vivo. Tuning ofCD3ζ-CAR-expression levels significantly improved thein vivoefficacy. Compared toTRAC-edited CAR-T cells, integration of a Her2-CAR intoCD3ζ conveyed similarin vitrotumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably,CD3ζ gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus,CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.Key pointsIntegration of ζ-deficient CARs intoCD3ζ gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf useCD3ζ-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions

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

“…Non-viral gene editing is a promising approach to create genetically-modified Tregs for therapeutic applications 9 due to economic advantages of non-viral vectors and reduced risk of insertional mutagenesis. Efficient non-viral knock-ins into Tregs has been reported previously with SpCas9 20,28,36,37 . The minimal transgene size of CD3ε-TruC strategy is advantageous, because linear dsDNA templates display significant dose-and also size-dependent toxicity in T cells 20,28,23,36 .…”

“…Previously, TRAC has been the gold-standard for knock-in of 2 nd generation CARs 25 . Miniaturization of the CAR transgene has been proposed by inserting truncated CD3ζ -deficient 2 nd -generation CARs into the CD3ζ gene in Tconv and Tregs 37 . Both approaches, TRAC 22 and CD3ζ 37 , eliminate TCR surface expression in Tregs and require adaption of the manufacturing to enrich redirected Tregs via constant antigen stimuli.…”

“…Miniaturization of the CAR transgene has been proposed by inserting truncated CD3ζ -deficient 2 nd -generation CARs into the CD3ζ gene in Tconv and Tregs 37 . Both approaches, TRAC 22 and CD3ζ 37 , eliminate TCR surface expression in Tregs and require adaption of the manufacturing to enrich redirected Tregs via constant antigen stimuli. Similar to our approach, SpCas9-mediated CD3ε gene editing was used to create TruC + Tconv with minimized transgenes 41,42 .…”

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

“…For example, efficiencies range from 3-17% for fluorescent proteins like GFP (1.5 kb insertion) and mCherry (2.1 kb insertion) transgenes, and less than 10% for a CAR (1.4 kb insertion). [21][22][23][24] Here, we describe a robust workflow using nonviral CRISPR-Cas9 ribonucleoproteins (RNP) to generate CAR NK cells against the diasialoganglioside GD2, an antigen expressed on a variety of solid tumors such as neuroblastoma, osteosarcoma and melanoma. 25 We identify several parameters influencing editing efficiencies to ensure efficient knock-out of the KLRC1 gene, encoding the inhibitory NKG2A receptor, and knock-in of an anti-GD2 CAR transgene at this locus.…”

“…For example, efficiencies range from 3-17% for fluorescent proteins like GFP (1.5 kb insertion) and mCherry (2.1 kb insertion) transgenes, and less than 10% for a CAR (1.4 kb insertion). 21–24…”