Modulation of TCR stimulation and pifithrin-α improve the genomic safety profile of CRISPR-engineered human T cells

Abstract

CRISPR-engineered chimeric antigen receptor (CAR) T cells are at the forefront of novel cancer treatments. However, several reports describe the occurrence of CRISPR-induced chromosomal aberrations. So far, measures to increase the genomic safety of T cell products focused mainly on the components of the CRISPR-Cas9 system and less on T cell-intrinsic features, such as their massive expansion after T cell receptor (TCR) stimulation. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with larger deletions. Editing of non-activated T cells reduces the risk of large deletions with the downside of reduced knockout efficiencies. Alternatively, the addition of the small-molecule pifithrin-α limits large deletions, chromosomal translocations, and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered T cells, including CAR T cells. Controlling T cell activation and pifithrin-α treatment are easily implementable strategies to improve the genomic integrity of CRISPR-engineered T cells.

Keywords: CAR T cell therapy; CRISPR engineering; T cell activation; aneuploidy; chromosomal aberrations; genomic integrity; human T cells; large deletions; pifithrin-alpha; translocations.

Graphical abstract

Figure 1

CRISPR-Cas9-induced deletion patterns in non-activated and activated human CD4 T cells (A) Schematic workflow of CRISPR-Cas9 editing experiments. (B) Percentage of indel formation at the AAVS1 gene locus determined by amplicon NGS sequencing. n(AAVS1) = 3, biological replicates. (C) Characterization of CD4 KO T cells. Left: representative flow cytometry plots of CD4 protein expression in AAVS1 KO control and CD4 KO T cells without (gray) or with TCR stimulation (red) after CRISPR-Cas9 editing. Right: quantification of CD4 expression and absolute numbers of CD4-negative cells in non-activated and activated CD4 T cells by flow cytometry analysis. n(CD4) = 3, biological replicates. (D) Flow cytometry analysis of PDCD1 KO T cells. Left: representative flow cytometry plots for PD-1 expression levels in PDCD1 KO and AAVS1 KO CD4 T cells (gray: non-activated T cells, red: activated T cells). Right: quantification of PD-1 expression levels and absolute cell numbers for PD-1-negative cells. n(PDCD1) = 3, biological replicates. (B–D) The respective means are indicated. Paired t test, ns: not significant, ∗p < 0.05, ∗∗p < 0.01. (E) Deletion sizes in CRISPR-Cas9-edited T cells at the AAVS1, CD4, or PDCD1 target loci in non-activated or activated T cells of the same donors (gray: non-activated; red: activated). Deletion patterns were analyzed by amplicon NGS sequencing. n(AAVS1, CD4, PDCD1) = 3, biological replicates.

Figure 2

Deletion sizes in CRISPR-Cas9-edited T cells correlate with cell proliferation speed (A) Representative example of flow cytometry gating strategy for the characterization and isolation of fast (blue) and slowly (green) proliferating CD4 T cells based on CFSE dilution pattern. Non-dividing cells were excluded from the analysis (gray peak on the right). (B) AAVS1 indel frequencies (NGS) and CD4 (flow cytometry) and PD-1 KO (flow cytometry) efficiencies in slowly and fast-dividing CD4 T cells based on CFSE dilution pattern. Paired t test, ns: not significant, ∗∗p < 0.01. (C) Quantification of deletion sizes dependent on the T cell proliferation speed based on amplicon NGS data. (B and C) n(AAVS1) = 3, n(CD4) = 3, n(PDCD1) = 4, biological replicates.

Figure 3

Deletion sizes after CRISPR-Cas9 editing in the presence of PFT-μ or PFT-α (A) NGS analysis of AAVS1 indel frequencies and flow cytometry analysis of CD4 and PD-1 KO efficiencies in activated CD4 T cells treated with DMSO or with PFT-μ. n(AAVS1) = 3, n(CD4) = 6, n(PDCD1) = 6, biological replicates, paired t test. (B) Percentages of AAVS1 modified NGS reads and CD4 and PD-1 protein levels of DMSO or PFT-α-treated activated CD4 T cells determined by flow cytometry. n(AAVS1) = 3, n(CD4) = 6, n(PDCD1) = 7, biological replicates, paired t test. (C) Representative histograms of CFSE dilution patterns in AAVS1, CD4, and PDCD1 KO T cells treated with PFT-μ/α. (D) Quantification of fast-proliferating (CFSE-low) cells in AAVS1, CD4, and PDCD1 KO T cells treated with PFT-μ/α. Cells were pre-gated on CD4- or PD-1-negative cells, respectively. Medians are indicated. Paired t test. (E) Detected deletion sizes by amplicon NGS in non-activated, fast or slowly dividing CRISPR-Cas9-edited T cells with DMSO or PFT-μ. n(AAVS1, CD4, PDCD1) = 3, biological replicates. Data correspond to (C) and (D). (F) Deletion sizes based on the activation and proliferation speed in CD4 T cells incubated with DMSO or PFT-α determined by amplicon NGS. Conditions with non-activated T cells and non-inhibitor-treated cells are the same as shown in Figure 2C. n(AAVS1) = 3, n(CD4) = 3, n(PDCD1) = 4, biological replicates. Data correspond to (C) and (D). (G and H) NGS quantification of AAVS1 (G) and TP53 KO rates (H) in AAVS1 and TP53 double-edited T cells after PFT-μ/α treatment. Cells were pre-sorted on CD4⁻ or PD-1-negative cells, respectively. Medians are indicated. Two-way ANOVA with Tukey’s multiple comparison test. (I and J) Amplicon NGS results of AAVS1_CD4, AAVS1_PDCD1, TP53_CD4, and TP53_PDCD1-edited T cells after the addition of PFT-μ/α. (G–J) n(CD4) = 4, n(PDCD1) = 5, biological replicates. ns: not significant, ∗p < 0.05, ∗∗∗p < 0.001.

Figure 4

Impact of PFT-α on CD4 T cell subsets TN, TEM, and TCM cells were flow purified, CFSE labeled, nucleofected with Cas9 RNPs, and treated with either DMSO or PFT-α. (A) Representative example of PD-1 expression levels in different CD4 T cell subsets of one donor with either AAVS1 or PDCD1 KO. PDCD1 KO T cells were treated with DMSO or PFT-α. The respective CFSE dilution pattern of each sample is depicted on the right. (B) PD-1 expression levels in AAVS1 KO and PDCD1 KO TN, TEM, and TCM cells. PDCD1 KO: n(TN) = 6, n(TEM) = 5, n(TCM) = 6; AAVS1 KO: n(TN) = 6, n(TCM) = 2, n(TEM) = 2, biological replicates. Means with SD are shown. (C) Percentage of modified PDCD1 reads with DMSO or PFT-α treatment. Paired t test. (D) Percentage of cell proliferation based on CD4-positive CFSE_low T cells in TN, TEM, and TCM cells with or without PFT-α treatment (gating: Figure S3). Paired t test. (E–G) Subset compositions of input TN (E), TEM (F), and TCM (G) cells after KO, TCR stimulation, and DMSO or PFT-α treatment (gated on TN-like, TEM, TCM, and TEMRA; gating strategy: Figure S5). Two-way ANOVA with Šídák’s multiple comparison test; no significant changes detected. (H–J) Aligned reads of fast and slow proliferating T cells in TN (H), TEM (I), and TCM (J) cells with and without PFT-α treatment. (E–J) n(TN) = 6, n(TEM) = 5, n(TCM) = 6, biological replicates. ns: not significant, ∗p < 0.05, ∗∗∗p < 0.001.

Figure 5

Functionality of CAR T cells engineered in the presence of PFT-α (Α) CD8 T cells were activated, nucleofected with NT-, AAVS1-, or TRAC-targeting Cas9 RNPs, transduced 48 h later with αCD19-CAR retrovirus, and treated with DMSO or PFT-α. KO rates of AAVS1 and TRAC KO CAR T cells with or without PFT-α treatment. Paired t test. (B) Deletion sizes in AAVS1 and TRAC KO cells with DMSO or PFT-α. (A and B) n = 6, biological replicates. (C) IFNγ-positive CAR T cells after 4 h in Nalm6 ffluc-GFP killing assay in different CAR T cell:tumor cell ratios. (D) Nalm6 ffluc-GFP cell count after 24 h co-culture with CAR T cells. (C and D) Killing assays were performed in technical duplicates. Means are indicated. n = 4, biological replicates. Results of DMSO and PFT-α conditions were normalized for differences in transduction rates. two-way ANOVA (mixed-effects model) with Tukey’s multiple comparison test; no significant changes were detected between the respective DMSO and PFT-α groups. (E) Outline of TRAC KO CAR T cell challenge in Nalm6 ffluc-GFP tumor model in NSGS mice. (F) Deletion sizes in TRAC KO CAR CD4 and CD8 T cells engineered in the presence of DMSO or PFT-α. n = 1. (G) Bioluminescence pictures of Nalm6 ffluc-GFP tumor burden at day 0, 6, and 9 of mock-treated mice and mice treated with TRAC KO CAR T cells with or without PFT-α. (H) Quantification of bioluminescence signal of mice shown in (G). Unspecific head signals were excluded from the analysis. Two-way ANOVA, mean with SD is shown. (I) Frequency of CAR-positive T cells in the peripheral blood at day 6. Median is depicted. (J) Nalm6 ffluc-GFP tumor cell counts in the peripheral blood at day 6 and 9. Mean with SD. (K) Nalm6 ffluc-GFP tumor cell counts in blood, spleen, and bone marrow at day 9 after CAR T cell injection. Median is shown. (F–K): n(mock) = 3, n(TRAC KO + DMSO, TRAC KO + PFT-α) = 5. ns: not significant, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Figure 6

Impact of PFT-α on CRISPR-induced chromosomal aberrations and aneuploidy (A) Chromosomal rearrangements. Circos plot visualization of chromosomal aberrations at the CD4 target locus in activated and non-activated T cells treated with or without PFT-α as detected by CAST-Seq. CD4: Chromosomal aberrations at on-target site. PDCD1: Chromosomal translocations to PDCD1 target locus. OMT: off-target-mediated translocation to RABL6 locus. (B) Representative example of CAST-seq read coverage plots of ± 5 kb around the CD4 on-target site (donor 2 is depicted). The x axis indicates the chromosomal coordinates, the y axis the log2 read count per million (CPM), and the dotted line the cleavage site. Deletions (DEL) are shown in orange, inversions (INV) in purple. (C) Percentage of large chromosomal aberrations. Fraction of CAST-seq reads with a distance of more than 200 bp from the cleavage site within a ± 5 kb window around the cleavage site. (D) Mean deletion lengths at the CD4 locus. (E) Quantification of chromosomal translocations to PDCD1 locus. Translocation reads were normalized to absolute read numbers. (C–E) One-tailed paired t test. (A–E) n = 3, biological replicates. (F) Chromosomal loss. Number of TRAC KO cells treated with or without PFT-α with CRISPR-induced chromosome 14 (chr14) haploidy or diploidy analyzed by scKaryo-seq (left). Percentage of chr14 haploid cells (right). Combined analysis of cells of two donors. Fisher’s exact test. ns: not significant, ∗p < 0.05.