SEED-Selection enables high-efficiency enrichment of primary T cells edited at multiple loci

Abstract

Engineering T cell specificity and function at multiple loci can generate more effective cellular therapies, but current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here we develop a one-step process to enrich unlabeled cells containing knock-ins at multiple target loci using a family of repair templates named synthetic exon expression disruptors (SEEDs). SEEDs associate transgene integration with the disruption of a paired target endogenous surface protein while preserving target expression in nonmodified and partially edited cells to enable their removal (SEED-Selection). We design SEEDs to modify three critical loci encoding T cell specificity, coreceptor expression and major histocompatibility complex expression. The results demonstrate up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). This method is compatible with existing clinical manufacturing workflows and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.

Extended Data Fig. 1:. Comparison of SEED-Selection to alternative enrichment strategies

Chart detailing different strategies for isolating cells engineered to express a CAR or other transgene. Representative HDRTs are shown for each selection strategy. *: STOP codon; BP: branchpoint; SA: splice acceptor; SD: splice donor; pA: polyA signal; LHA/RHA: homology arms.

Extended Data Figure 2:. Intronic editing sites preserve target gene expression

a, Panel of gRNAs targeting TRAC exon (e1) and TRAC intron (i2–i10); not depicted to scale. b, Assessment of TCR expression by flow cytometry and indel generation by bulk genomic DNA sequencing in T cells after editing with gRNAs depicted in b (n = 2 donors). c, Panel of gRNAs targeting B2M introns; not depicted to scale. d, Assessment of B2M expression by flow cytometry and indel generation by bulk genomic DNA sequencing in T cells after editing with gRNAs depicted in c (n = 2 donors).

Extended Data Figure 3:. Evaluation of TRAC intron and exon targeted gRNAs

a, Percentage of TCR^– CAR^– cells after editing with TRAC RNP alone or RNP with CAR HDRT (n = 3 donors). Data displayed as mean plus SEM. Significance was assessed with a two-sided paired t test. b, Comparison of TRAC-intron and exon-targeted HDRTs encoding a CAR and EGFRt. c–g, T cells were edited with TRAC intron or TRAC exon-targeted RNPs and HDRTs (b). Edited cells from each condition were then immunomagnetically purified with anti-TCR (n = 2 donors). c, Flow cytometry plots of TCR and CAR (anti-mouse F(ab’)₂) expression d, Histograms of CAR expression in TCR^– CAR⁺ cells. e, Median fluorescence intensity (MFI) of CAR expression in TCR^– CAR⁺ cells. f, Percentage of TCR^– CAR⁺ cells in purified (blue) and non-purified samples (grey). g, Relative enrichment of fully edited cells after purification: (% of purified sample) / (% of non-purified sample).

Extended Data Figure 4:. Recovery of edited cells following SEED-Selection

a, Estimated percentage of target cell population recovered following SEED-Selection. b, Input and output of total cells and target cells for SEED-Selection. Purifications for each target-payload purification were performed separately. Dots represent data from biological replicates within an experiment (n = 2 donors for TRAC-CAR/B2M-CD47 double KI purification and TRAC-NY-ESO/B2M-CD47/CD4-CD8 triple KI purification; n = 3 donors for all other conditions) Additional yield data is included in Supplementary Table 1.

Extended Data Figure 5:. Optimization and characterization of B2M targeted SEEDs

a–e, T cells were edited with RNP targeting B2M and transduced with varying MOIs of AAV encoding CD47. In indicated conditions, editing was performed in the presence of M3814 (n = 3 donors). Cells transduced at an MOI of 1 × 10⁴ and edited in the presence of M3814 shown in select panels (a,b). a, Representative flow cytometry plots of B2M and CD47 expression in edited and non-edited cells. b, Median fluorescence intensity (MFI) of B2M or CD47 expression in subpopulations of edited cells. Data displayed as mean plus SEM. Significance was assessed using a repeated-measures one-way ANOVA and Turkey’s multiple comparison test. c, Percentage of CD47⁺ cells, d, Percentage of B2M^– CD47⁺ cells, e, Percentage of CD47⁺ cells with full B2M disruption. Data for c–e displayed as mean plus SEM. f,g, T cells edited with RNP targeting B2M (+M3814) and transduced with high (3 × 10⁵) or low (1 × 10⁴) MOIs of B2M-CD47 SEED HDRT AAV were immunomagnetically purified with anti-B2M (n = 3 donors). f, Representative flow cytometry plots of B2M and CD47 expression. g, Percentage of B2M^– CD47⁺ cells. Data displayed as mean plus SEM (n = 3 donors). h, Representative flow cytometry plots of B2M and CD47 expression used to evaluate CD47+:CD47 ratio in Fig. 1h.

Extended Data Figure 6:. Efficient low-MOI editing can be achieved with high density transductions

a, Comparison of high and low MOI editing protocols. b–d, T cells were edited with RNPs targeting TRAC and B2M (+M3814) and transduced with TRAC-CAR and B2M-CD47 SEED HDRTs in a G-REX plate at an MOI of 3 × 10⁴ (per AAV). Edited cells were then immunomagnetically purified with anti-TCR and anti-B2M (n = 3 donors). b, Representative flow cytometry plots of TCR, CAR, B2M, and CD47 expression. c, Percentage of B2M⁺ or TCR⁺ cells and fully edited cells (TCR^– CAR⁺ B2M^– CD47⁺). d, Number of cells generated from editing 4 × 10⁶ cells followed by a 10-day expansion.

Extended Data Figure 7:. Characterization of individual and pooled libraries of epitope-edited HITs

a, Flow cytometry plots of BW242 binding and HIT expression (mouse F(ab’)₂) in T cells edited with either a HIT β102 or β116 saturation mutagenesis pool. Boxes indicate sorted populations. b, Relative enrichment of mutations in HIT⁺ BW242^– cells versus HIT⁺ BW242⁺ in cells from a. Each dot represents enrichment for a single codon. Bars represent the average enrichment of all codons for an amino acid (n = 1 donor). c, Representative flow cytometry plots of BW242 binding and HIT expression in T cells individually edited with TRAC-exon targeted HDRTs encoding HIT receptor variants. Median fluorescence intensity (MFI) values for BW242 binding (for Fig. 2j) were determined based on the HIT⁺ gated population (for transduced samples) or the TCR KO gated population (n = 3 donors). d, Representative flow cytometry plots of CD3 and HIT expression in T cells individually edited with TRAC-exon targeted HDRTs encoding HIT receptor variants (n = 3 donors), e, (continued from Fig. 3n) Cytotoxic activity of T cells with a non-modified HIT (blue), epitope-edited HIT (red), or TCR knockout (grey) against Nalm6 lines (technical triplicate, data shown from 1 of 4 donors). Data displayed as mean plus SEM.

Extended Data Figure 8:. Validation of epitope editing in transgenic TCRs

a, Flow cytometry plots quantifying CD3 expression and NY-ESO-1 dextramer binding in CD8⁺ cells. Editing was performed with RNPs targeting TRAC or TRAC/TRBC and HDRTs encoding non-modified (1G4-LY^WT) or epitope-edited (1G4-LY^112K) versions of 1G4-LY (n = 2 donors). b, Flow cytometry plots quantifying BW242 binding and MART-1 dextramer binding in CD8⁺ cells. Editing was performed with RNPs targeting TRAC or TRAC/TRBC and an HDRT encoding an epitope-edited version of a MART-1 TCR (DMF5) (n = 1 donor). c, Flow cytometry plots of BW242 binding and MART-1 dextramer binding in CD8⁺–gated T cells edited with a MART-1 TCR HDRT after immunomagnetic BW242 depletion (n = 1 donor).

Extended Data Figure 9:. SEED-Selection facilitates co-receptor swapping

a, BW242 binding and NY-ESO-1 dextramer binding in CD4⁺ and CD8⁺ cells edited with a 1G4-LY^112K SEED. b, Assessment of CD4 expression by flow cytometry and indel generation by bulk genomic DNA sequencing in T cells after editing with intron-targeted gRNAs (n = 2 donors). c, Expression of CD4 or CD8 in subpopulations of non-purified CD8 SEED edited cells. MFI: median fluorescence intensity. Data displayed as mean plus SEM (n = 3 donors). d, Assessment of cytotoxic activity of CD4⁺ T cells against NY-ESO-1⁺ A375 target cells (continued from Fig. 5h), Non-edited T cells (dark gray), 1G4-LY SEED edited (red), 1G4-LY SEED and CD8 SEED edited (blue). Data displayed as mean (dark line) plus SEM (shaded area) (technical triplicate, n = 3). Significance was assessed at 72 hours using a repeated-measures one-way ANOVA and Turkey’s multiple comparison test.

Extended Data Figure 10:. Optimized multi-locus low-MOI editing at TRAC, B2M, and CD4

CD4⁺ T cells were edited with RNPs targeting TRAC, B2M, and CD4 (+M3814) and transduced with SEEDs encoding 1G4-LY, CD8, and CD47 in a G-REX plate at an MOI of 3 × 10⁴ (per AAV). Edited cells were then immunomagnetically purified with BW242, anti-CD4, and anti-B2M (n = 3 donors). a, Flow cytometry plots of SEED target expression (endogenous TCR, CD4, B2M), SEED payload expression (CD8, CD47), and NY-ESO-1 dextramer binding. b, Percentage of cells expressing any SEED target or a mispaired TCR (BW242⁺ or CD4⁺ or B2M⁺) or triple knockout/triple knock-in cells with correct 1G4-LY pairing (BW242^– B2M^– CD4^– Dextramer⁺ CD47⁺ CD8⁺). c, Number of cells generated from editing 4 × 10⁶ cells followed by a 9-day expansion.

Figure 1:. SEEDs couple transgene integration to the target protein disruption

a, Overview of editing outcomes generated with an intron-targeted gRNA and a SEED HDRT. Immunomagnetic reagents are used to deplete cells that retain expression of the surface protein targeted by a SEED, thereby enriching for cells with transgene integration. b, Diagram of a TRAC intron-targeted SEED HDRT encoding a CAR and EGFRt. c,d, T cells were edited with TRAC intron-targeted RNP and HDRT (b) then immunomagnetically purified with anti-TCR (n = 3 donors). c, Flow cytometry plots of TCR and CAR expression (anti-mouse F(ab’)₂). d, Percentage of TCR⁺ and TCR^– CAR⁺ cells. e, Diagram of a B2M intron-targeted SEED HDRT encoding CD47. f–h, T cells were edited with B2M intron-targeted RNP and HDRT (e) in the presence of M3814 then immunomagnetically purified with anti-B2M (n = 3 donors). f, Flow cytometry plots of B2M and CD47 expression. g, Percentage of B2M⁺ and B2M^– CD47⁺ cells. h, Genomic DNA PCR targeting the SEED integration site at B2M. Amplicon for non-edited alleles (black triangle); amplicon for HDRT integration (blue triangle) (n = 3 donors). i, Fold enrichment of CD47⁺ SEED-edited cells relative to CD47^– cells after co-culture with NK cells (n = 3 T cell donors). Data displayed as mean plus SEM. Significance was assessed with a two-way ANOVA and Šidák’s multiple comparisons test. BP: branchpoint; SA: splice acceptor; SD: splice donor; LHA/RHA: homology arms; SEM: Standard Error of the Mean.

Fig 2:. SEED-Selection can simultaneously enrich for edits at multiple loci

a–c, T cells were edited with TRAC and B2M RNPs (+M3814) and transduced with TRAC-CAR SEED and B2M-CD47 SEED HDRTs. Edited cells were then immunomagnetically purified with anti-B2M and anti-TCR (n = 2 donors). a, Multiplexed editing and enrichment strategy. b, Representative flow cytometry plots of TCR, CAR, B2M, and CD47 expression. c, Percentage of B2M⁺ or TCR⁺ cells and fully edited cells (TCR^– CAR⁺ B2M^– CD47⁺). d–g, 1.6 × 10⁸ T cells were edited with TRAC and B2M RNPs and transduced with TRAC-CAR SEED and B2M-CD47 SEED HDRTs using a GMP-compatible workflow (n = 2 donors). d, Overview of large-scale editing workflow. e, Expansion of cells after multiplexed editing. f, Number and composition of edited cells after expansion. g, Representative flow cytometry plots of TCR, CAR, B2M, and CD47 expression.

Figure 3:. Epitope editing allows for TCR-based receptors to be used in TCR SEEDs

a, Diagram of a TCR and a HIT. b, Diagram of a TRAC exon-targeted HDRT encoding a HIT. c, Flow cytometry of anti-TCR (BW242) binding and HIT expression (anti-mouse F(ab’)₂) in T cells with a HIT at TRAC (n = 1 donor). d, Screening workflow for epitope mapping. e–g, T cells were edited to express a library of mutated HITs then purified with BW242. BW242 binding and HIT expression were quantified with flow cytometry before (e) and after (f) purification. HIT⁺ BW242^– cells were sorted (blue box) for sequencing (n = 1 donor). g, Enrichment of mutations in HIT⁺ BW242^– cells (blue box in f) compared to the original library (e). Dots represent the average of tested codons for an amino acid. h, Flow cytometry of BW242 binding and HIT expression in T cells edited with a HIT β112 saturation mutagenesis pool. Boxes indicate sorted populations. I, Enrichment of mutations in HIT⁺ BW242^– cells versus HIT⁺ BW242⁺ cells. Dots represents enrichment for a single codon (n = 1 donor). Lines display the average enrichment for an amino acid. J, BW242 binding for HIT⁺ T cells edited with HIT variants (n = 3 donors) compared with average enrichment scores (from i). Black line indicates BW242 MFI for TCR^– cells. MFI SEM displayed by bars (HIT mutants) or shaded grey area (TCR^–). k, Diagram of a TRAC SEED HDRT encoding an epitope-edited HIT. l,m, T cells were edited with a HIT SEED (+M3814) and then purified with BW242 (n = 3 donors) l, Flow cytometry of BW242 binding and HIT expression m, Percentage of BW242⁺ and BW242⁻ HIT⁺ cells. n, Cytotoxic activity of T cells with a non-modified HIT (blue), epitope-edited HIT (red), or TCR knockout (grey) against Nalm6 lines (technical triplicate, data shown from 1 of 4 donors). Histograms show Nalm6 CD19 expression (flow cytometry). Unstained cells shown in grey. Data displayed as mean plus SEM. BP: branchpoint; SA: splice acceptor; SD: splice donor; LHA/RHA: homology arms; MFI: median fluorescence intensity; SEM: Standard Error of the Mean.

Figure 4:. SEED-Selection depletes TRAC-edited TCR-swapped cells that express mispaired TCRs

a, Schematic of possible TCR pairs in non-edited and TRAC-edited T cells engineered to express the 1G4-LY^112K TCR (specific for NY-ESO-1). b, Diagram of a TRAC exon-targeted HDRT encoding 1G4-LY. c, Flow cytometry plots of BW242 binding and NY-ESO-1 dextramer binding in CD8⁺ cells. Editing was performed with RNP(s) targeting TRAC or TRAC/TRBC and HDRTs (a) encoding non-modified (1G4-LY^WT) or epitope-edited (1G4-LY^112K) versions of 1G4-LY (n = 2 donors). d, Diagram of a TRAC intron-targeted SEED HDRT encoding 1G4-LY. e,f, T cells were edited with a 1G4-LY SEED (+M3814) (d) and immunomagnetically purified with BW242 (n = 3 donors). e, Flow cytometry plots of BW242 binding and NY-ESO-1 dextramer binding for cells gated on CD8⁺. f, Percentage of cells with an endogenous or mispaired TCRs (BW242⁺) or correctly paired 1G4-LY (Dextramer⁺ BW242⁻). BP: branchpoint; SA: splice acceptor; SD: splice donor; LHA/RHA: homology arms.

Figure 5:. SEED-Selection enables the enrichment of TCR-swapped, co-receptor-swapped cells

a, Diagram of a CD4 intron-targeted SEED HDRT encoding CD8α/β. b,c, CD4⁺ T cells were edited with a CD8 SEED (a) (+M3814) and immunomagnetically purified with anti-CD4 (n = 3 donors). b, Flow cytometry plots of CD4 and CD8 expression. c, Percentage of CD4⁺ and CD4^– CD8⁺ cells. d–h, CD4⁺ T cells were edited with 1G4-LY and CD8 SEEDs (+M3814) and immunomagnetically purified with BW242 and anti-CD4 (n = 3 donors). d, Overview of editing and selection strategy. e, Flow cytometry plots of CD4 and CD8 expression and BW242 and NY-ESO-1 dextramer binding. f, Percentage of cells which express CD4 or an endogenous or mispaired TCR (BW242⁺); Percentage of fully-edited cells with minimal mispairing (BW242^– CD4^– Dextramer⁺ CD8⁺). g, Assessment of NY-ESO-1 dextramer binding by flow cytometry in CD4⁺ CD8^– BW242^– 1G4-LY⁺ cells (red), CD4^– CD8⁺ BW242^– 1G4-LY⁺ cells (blue), and non-edited CD4⁺ T cells (grey) (n = 3 donors). Data displayed as mean plus SEM. h, Assessment of cytotoxic activity of CD4⁺ T cells against NY-ESO-1⁺ A375 target cells (technical triplicate, data shown from 1 of 3 donors). Non-edited T cells (dark gray), 1G4-LY SEED edited (red), 1G4-LY SEED and CD8 SEED edited (blue). Data displayed as mean (bold line) plus SEM (shaded area). Significance was assessed at 72 hours. Significance for g and h was assessed using a repeated-measures one-way ANOVA and Turkey’s multiple comparison test. BP: branchpoint; SA: splice acceptor; SD: splice donor; LHA/RHA: homology arms; MFI: median fluorescence intensity; SEM: Standard Error of the Mean.

Figure 6:. SEED-Selection enables the isolation of complex cell therapies without enriching for translocations or chromosomal loss between target loci

a–c, CD4⁺ T cells were edited with RNPs targeting TRAC, B2M, and CD4 (+M3814) and transduced with SEEDs encoding 1G4-LY, CD8, and CD47, respectively. Edited cells were then immunomagnetically purified with BW242, anti-CD4, and anti-B2M (n = 2 donors). a, Diagram of the workflow for multiplexed editing and enrichment. b, Flow cytometry plots of SEED target expression (endogenous TCR, CD4, B2M), SEED payload expression (CD8, CD47), and NY-ESO-1 dextramer binding. c, Percentage of cells expressing any SEED target or a mispaired TCR (BW242⁺ or CD4⁺ or B2M⁺) or triple knockout/triple knock-in cells with correct 1G4-LY pairing (BW242^– B2M^– CD4^– Dextramer⁺ CD47⁺ CD8⁺). d, Assessment of balanced translocations between TRAC, B2M, and CD4 after multiplexed editing (+M3814) and SEED-Selection via digital droplet PCR after a 9-day expansion (n = 3 donors). e–g, Partial and total chromosome loss was assessed in after multiplexed editing and SEED-Selection via single cell RNA sequencing (n = 3 donors). e, Representative gene expression profiles of cells with partial or complete loss of chromosome 14 after multiplexed editing. f, Normalized frequency of cells with partial and total chromosomal loss. g, Normalized frequency of cells with 1 or more chromosomal abnormalities. Significance for d, f, and g was assessed using repeated-measures one-way ANOVAs and Turkey’s multiple comparison test.