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High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition

Nature Biotechnology (2023)Cite this article

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Abstract

Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.

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Fig. 1: AZD7648 is the most potent DNA-PKcs inhibitor for improving gene targeting in PSCs.
Fig. 2: AZD7648 improves gene targeting in HSPCs.
Fig. 3: AZD7648 treatment improves gene targeting with seemingly inactive and low-activity gRNAs.
Fig. 4: AZD7648 improves gene targeting with lower amounts of RNP and AAV6.
Fig. 5: AZD7648 improves gene targeting in primary human T cells and HBECs.

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Data availability

NGS data for off-target analysis have been deposited in the National Center for Biotechnology Informationʼs Sequence Read Archive database (accession number PRJNA982854)87 and can be accessed at http://www.ncbi.nlm.nih.gov/bioproject/982854. Sequences of the gRNA, PCR primers, ddPCR primers and probes have been included in Supplementary Table 1. Raw data for ICE analysis and screenshots of Sanger sequencing chromatograms are provided in the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank the Binns Program for Cord Blood Research for providing cord blood CD34+ HSPCs. We thank the FACS Core Facility at the Stanford Institute of Stem Cell Biology and Regenerative Medicine for access to the FACS machines. We would like to thank the Taube and Koret Foundation for funding support. We thank F. Suchy and the laboratory of H. Nakauchi at Stanford University for help with ddPCR primer/probe design.

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Authors and Affiliations

  1. Department of Pediatrics, Stanford University, Stanford, CA, USA

    Sridhar Selvaraj, William N. Feist, Sebastien Viel, Sriram Vaidyanathan, Amanda M. Dudek, Marc Gastou, Sarah J. Rockwood, Freja K. Ekman, Aluya R. Oseghale, Liwen Xu, Mara Pavel-Dinu, Sofia E. Luna, M. Kyle Cromer, Ruhi Sayana, Natalia Gomez-Ospina & Matthew H. Porteus

  2. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA

    Sridhar Selvaraj, William N. Feist, Sebastien Viel, Sriram Vaidyanathan, Amanda M. Dudek, Marc Gastou, Sarah J. Rockwood, Freja K. Ekman, Aluya R. Oseghale, Liwen Xu, Mara Pavel-Dinu, Sofia E. Luna, M. Kyle Cromer, Ruhi Sayana, Natalia Gomez-Ospina & Matthew H. Porteus

  3. Immunology Department, Lyon Sud University Hospital, Pierre-Bénite, France

    Sebastien Viel

  4. International Center of Research in Infectiology, Lyon University, INSERM U1111, CNRS UMR 5308, ENS, UCBL, Lyon, France

    Sebastien Viel

  5. Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA

    Sriram Vaidyanathan

  6. Department of Pediatrics, The Ohio State University, Columbus, OH, USA

    Sriram Vaidyanathan

  7. Department of Surgery, University of California, San Francisco, San Francisco, CA, USA

    M. Kyle Cromer

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  1. Sridhar Selvaraj
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Contributions

S.S., W.N.F., S. Viel, S. Vaidyanathan, A.M.D., M.G., S.J.R., F.K.E., A.R.O., L.X., M.P.-D. and S.E.L. designed and performed experiments and analyzed data. M.K.C. provided reagents for experiments. R.S. performed experiments and analyzed data. N.G.-O. and M.H.P. supervised the project. S.S. and M.H.P wrote the initial and final drafts of the manuscript with input from other authors.

Corresponding author

Correspondence to Matthew H. Porteus.

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Competing interests

M.H.P. is a member of the scientific advisory board of Allogene Therapeutics and a member of the board of directors of Graphite Bio. S.S. and M.H.P. are inventors on intellectual property related to this work.

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Extended data

Extended Data Fig. 1 Comparison of different DNA-PKcs inhibitors for gene targeting with AZD7648.

a. Allelic gene targeting efficiency post gene editing at the CCR5 locus for introduction of two stop codons with different concentrations of DNA-PKcs inhibitors (AZD7648, KU57788, LTURM34 and BAY8400) in comparison with the untreated cells (UNT) as measured by ICE analysis at 4 days post-gene editing (n = 3). b. Allelic gene targeting efficiency at the CCR5 locus for knock-in of UBC-GFP-bGHpA sequence with two different concentrations (0.5 and 0.25 μM) of various DNA-PKcs inhibitors (AZD7648, M3814, VX984 and BAY8400) as measured by ddPCR analysis at 3 days post-gene editing (n = 3). c. MTT cell viability assay on gene targeted PSCs (b) at 24 h, 48 h and 72 h post-gene editing represented as percent cell viability normalized to the mock cells (n = 3). All data in a, b and c are shown as mean ± SEM. Data in a, b and c were compared with one-way ANOVA and Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns denotes not significant.

Source data

Extended Data Fig. 2 Biochemical validation of DNA-PKcs inhibition and CCR5 gene targeting with AZD7648 in PSC.

a. Western blot analysis for phospho DNA-PKcs (pSer2056), DNA-PKcs and phospho AKT (pSer473) in PSCs treated with bleomycin ± AZD for 2 h or gene targeted with and without AZD treatment at 2 h post gene editing. ACTB is used as a loading control. Control denotes sample not treated with bleomycin. Mock denotes control sample nucleofected without RNP. UNT denotes sample either treated with bleomycin or RNP-AAV6 gene editing and AZD denotes AZD7648 treated sample with either bleomycin or RNP-AAV6 gene editing treatment as indicated. b. Allelic gene targeting efficiency of PSCs gene targeted at the CCR5 locus for knock-in of UBC-GFP-bGHpA sequence (a) with and without AZD treatment at 72 h post gene editing as measured by ddPCR (n = 1). c. Percentage of gene targeted cells at the CCR5 locus for knock-in of UBC-GFP-bGHpA sequence with two different concentrations of AZD7648 (0.5 and 0.25 μM) in comparison with the untreated cells (UNT) as measured by flow cytometry for GFP at 5-days post-gene editing (n = 3). Mock and RNP only cells were used as negative controls. d. Allelic distribution of WT, INDEL, HDR frequencies in CCR5 gene edited PSCs (c) (n = 3). HDR frequency was measured by ddPCR analysis, WT and INDEL frequencies were measured using ICE analysis. Mean HDR to INDEL ratio is represented above the bars. All data in c and d are shown as mean ± SEM.

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Extended Data Fig. 3 Pluripotency, trilineage differentiation and single cell cloning analysis of CCR5 gene targeted PSCs.

a. Percentage of cells positive for various markers of pluripotency (SSEA4, OCT3/4, SOX2 and NANOG) in the CCR5 gene edited PSCs (n = 3). SSEA4 expression was measured by flow cytometry analysis. OCT3/4, SOX2 and NANOG expression was assessed by quantification of immunofluorescence staining images for corresponding markers in fixed PSCs and normalized to the total cell count measured through DAPI staining (n = 3). All data are shown as mean ± SEM. b. CCR5 gene edited PSCs were differentiated into the three germ layers. Mean frequency of differentiated cells as assessed by flow cytometry for the expression of corresponding markers for ectoderm (PAX6 and NES), mesoderm (CD56 and T) and endoderm (CXCR4 and SOX17) (n = 2). c. PSCs were gene targeted at the CCR5 locus with and without different concentrations of AZD7648 (0.5, 0.25 and 0.1 µM) for single cell cloning analysis. Allelic gene targeting efficiency was measured using ddPCR analysis (n = 1). d. Gene targeted PSCs (c) were subjected to single cell cloning and the frequency of clones with mono-, bi-allelic and no gene targeting was measured using ddPCR and PCR analysis. For each condition, 9-10 clones were picked and analyzed (n = 1). e. Mean allelic gene targeting efficiency in PSC at the CCR5 locus for the knock-in of UBC-GFP-bGHpA sequence with pre-treatment only (preAZD-UNT), post-treatment (AZD) only and pre+post treatment (pre+post AZD) with AZD7648 (0.5 µM) (n = 2). For pre-treatment, cells were treated with AZD7648 for 24 hours before gene targeting and for post-treatment, cells were treated with AZD7648 for 24 hours post gene editing.

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Extended Data Fig. 4 AZD7648 treatment improves gene targeting at HBB, CFTR and HBA1 loci in PSC.

a. Schematic for gene targeting at the HBB locus to edit SCD (E6V) mutation in exon 1 using RNP/AAV6 gene editing (upper panel). Allelic distribution of WT, INDEL and HDR frequencies following gene editing at the HBB locus with and without AZD7648 treatment as measured by ICE analysis (lower panel) (n = 3). Mean HDR to INDEL ratio is represented above the bars. b. Schematic for gene targeting at the CFTR locus to edit CF disease mutation (ΔF508) in exon 11 using RNP/AAV6 gene editing (upper panel). Allelic distribution of WT, INDEL and HDR frequencies following gene editing with and without AZD7648 treatment at the CFTR locus as measured by ICE analysis (lower panel) (n = 3). Mean HDR to INDEL ratio is represented above the bars. c. Schematic for gene targeting at the HBB locus for knock-in of UBC-GFP-bGHpA sequence using RNP/AAV6 gene editing (upper panel). Allelic distribution of WT, INDEL and HDR frequencies following gene editing at the HBB locus with and without AZD7648 and M3814 (2 and 0.5 μM) treatment (lower panel) (n = 3). HDR efficiency was measured through ddPCR analysis. WT and INDEL frequencies were measured by ICE analysis. Mean HDR to INDEL ratio is represented above the bars. d. Schematic for gene replacement at the HBA1 locus for knock-in of transgene-2A-YFP sequence using RNP/AAV6 gene editing (upper panel). Allelic distribution of WT, INDEL and HDR frequencies following gene editing at the HBA1 locus with and without AZD7648 and M3814 (2 and 0.5 μM) treatment (lower panel) (n = 3). HDR efficiency was measured through ddPCR analysis. WT and INDEL frequencies were measured by ICE analysis. Mean HDR to INDEL ratio is represented above the bars. All data in a, b, c and d are shown as mean ± SEM.

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Extended Data Fig. 5 Gene targeting in HSPCs at CCR5 and HBA1 loci with AZD7648 treatment.

a. Comparison of different DNA-PKcs inhibitors for gene targeting at the CCR5 locus for knock-in of UBC-GFP-bGHpA sequence at two different concentrations (2 and 0.5 µM) in HSPCs. Allelic gene targeting efficiency was measured by ddPCR analysis and cell viability was assessed by measuring live cell count and normalizing the number to the Mock sample (n = 3). All data are shown as mean ± SEM. b. Allelic distribution of WT, INDEL and HDR frequencies following gene editing at the CCR5 locus for knock-in of UBC-GFP-bGHpA sequence with different concentrations of AZD7648 as indicated (n = 1). HDR frequency was measured using ddPCR, WT and INDEL frequencies were measured using ICE analysis. c. CFU assay was performed on Mock, RNP, AAV6 and RNP + AAV6 treated HSPCs following gene editing at the CCR5 locus with or without AZD7648 (0.5 μM) treatment. Plot shows the distribution of the mean percentages of CFU-GEMM (multi-potent granulocyte, erythroid, macrophage, megakaryocyte progenitor cells), CFU-GM (colony forming unit-granulocytes and monocytes) and BFU-E (erythroid burst forming units) colonies (n = 2). d. Schematic for gene replacement at the HBA1 locus to replace HBA1 with HBB sequence using RNP/AAV6 gene editing. e. Allelic distribution of WT, INDEL and HDR frequencies following gene editing at the HBA1 locus with or without AZD7648 treatment (0.5 μM) (n = 3). HDR efficiency was measured through ddPCR analysis. WT and INDEL frequencies were measured by ICE analysis. Mean HDR to INDEL ratio is represented above the bars. All data are shown as mean ± SEM. f. Mean percentage of viable cell count of HSPCs at 3 days post-gene targeting (GT) at CCR5 (n = 4), HBB (n = 2) and HBA1 (n = 2) loci in untreated (UNT) and AZD7648 (AZD, 0.5 μM) treated cells. Mock, AAV only and RNP only treated cells were included as controls. Viable cell counts were measured at 72 h post-gene editing and plotted as percentage relative to the mock cell count. For CCR5 gene targeting, cell viability is shown as mean ± SEM.

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Extended Data Fig. 6 Off-target analysis, gene targeting at CCR5 and HBB loci in HSPCs.

a. HSPCs gene targeted at HBB, CCR5 and HBA1 loci with and without AZD7648 treatment (0.5 μM) were assessed for off-target activity at the top off-target sites (OT1 for HBB, OT39 for CCR5 and OT1 for HBA1) through next generation sequencing (NGS). Mean frequency of reads with insertion and deletion INDELs is shown following SNP/INDEL detection analysis of the NGS data (n = 2). b. HSPCs were gene edited with Cas9 RNP at the CCR5 locus with and without AZD7648 treatment in the presence or absence of AAV6 donor template (for knock-in of two stop codons). Allelic distribution of mean frequencies of WT, insertions, deletions and HDR were determined by ICE analysis (n = 2). c. HSPCs were gene edited with Cas9 RNP at the HBB locus with and without AZD7648 treatment in the presence or absence of AAV6 donor template (for SCD mutation editing). Allelic distribution of frequencies of WT, insertions, deletions and HDR were determined by using ICE analysis (n = 3). All data are shown as mean ± SEM. d. Allelic distribution of mean WT, INDEL and HDR frequencies following gene editing at the HBB locus for editing SCD mutation using RNP and ssODN donor with and without AZD7648 treatment (0.5 μM) (n = 2). ssODN donor was tested at two different concentrations as indicated (2.5 and 5 μM).

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Extended Data Fig. 7 Gene targeting at the CCR5 and STING1 loci in HSPCs using seemingly low activity gRNAs.

a. HSPCs were gene targeted at the CCR5 locus for knock-in of UBC-GFP-bGH-pA sequence using high activity (sg11) and low activity gRNAs (sg1 and 4). Allelic distribution of WT, INDEL and HDR frequencies following gene editing at the CCR5 locus with and without AZD7648 treatment (n = 3). HDR frequency was determined by ddPCR analysis. WT and INDEL frequencies were determined by ICE analysis. Mean HDR to INDEL ratio is represented above the bars. All data are shown as mean ± SEM. b. Schematic for gene targeting at the IL2RG locus for knock-in of codon-optimized cDNA and bGHpA in exon 1 using RNP/AAV6 gene editing with a high activity gRNA (sg1) and a low activity gRNA (sg6). c. Schematic for gene targeting at the IL2RG locus for knock-in of codon-optimized cDNA and bGHpA in exon 1 using RNP/AAV6 gene editing with a high activity gRNA (sg1) and two low activity gRNAs (sg5, 7). d. Allelic distribution of WT, INDEL and HDR frequencies in HSPCs gene targeted at the IL2RG locus with or without AZD7648 treatment (0.5 μM) using sg1 and 6 gRNAs (n = 3) (b). HDR efficiency was measured through ddPCR analysis. WT and INDEL frequencies were measured by ICE analysis. Mean HDR to INDEL ratio is represented above the bars. All data are shown as mean ± SEM. e. Allelic distribution of mean WT, INDEL and HDR frequencies in HSPCs gene targeted at the IL2RG locus with or without AZD7648 treatment (0.5 μM) using sg1, 5 and 7 (n = 2) (c). HDR frequency was measured through ddPCR analysis. WT and INDEL frequencies were measured by ICE analysis. Mean HDR to INDEL ratio is represented above the bars.

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Extended Data Fig. 8 AZD7648 improves gene targeting with lower amounts of RNP/AAV6 in PSC and HSPC.

a. Frequency of gene targeted cells measured by flow cytometry for GFP following gene editing at the CCR5 locus for knock-in of UBC-GFP-bGH-pA sequence with or without AZD7648 treatment (0.25 μM) using varying amounts of Cas9-RNP as indicated and fixed amount of AAV6 donor (MOI:2500) in PSCs (n = 3). RNP-1X denotes 250 μg/ml of Cas9 protein complexed with 100 μg/ml of gRNA. b. Frequency of gene targeted cells measured by flow cytometry for GFP following gene editing at the CCR5 locus for knock-in of UBC-GFP-bGH-pA sequence in PSCs with or without AZD7648 treatment (0.25 μM) using a fixed amount of RNP and varying amounts of AAV6 donor (n = 3). c. Frequency of gene targeted cells measured by flow cytometry for GFP following gene editing at the CCR5 locus for knock-in of UBC-GFP-bGH-pA sequence in HSPCs with or without AZD7648 treatment (0.5 μM) using a fixed amount of RNP and varying amounts of AAV6 donor (n = 3). d. Frequency of gene targeted alleles measured by ICE analysis following gene editing at the HBB locus for editing the SCD mutation in HSPCs with or without AZD7648 treatment (0.5 μM) using a fixed amount of RNP and varying amounts of AAV6 donor (n = 3). e. Fold change in live cell count at D2, 4 and 6 post gene editing relative to D0 of HSPCs gene targeted at the HBB locus (d) (n = 3). f. p21 (CDKN1A) expression levels in gene targeted HSPCs (d) at D2, 4 and 6 post gene editing represented as values relative to non-gene edited control cells (n = 3). p21 expression levels were measured by RT-ddPCR and normalized to the levels of the house keeping gene, TBP. g. Number of AAV genomes in gene targeted HSPCs (d) at D2, 4 and 6 post gene editing represented as viral genomes (vg) per cell (n = 3). AAV genomes were quantified using ddPCR amplifying the AAV ITR and the values were normalized to a reference locus, ZEB2. Mean values are represented above the bars. All data are shown as mean ± SEM.

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Extended Data Fig. 9 AZD7648 improves gene targeting in T cells.

a. Comparison of different DNA-PKcs inhibitors for gene targeting at the CCR5 locus for knock-in of UBC-GFP-bGHpA sequence at two different concentrations (2 and 0.5 µM) in T cells. Allelic gene targeting efficiency was measured by ddPCR analysis and cell viability was assessed by measuring live cell counts and normalizing the number to the Mock sample (n = 3). b. Frequency of gene targeted cells as measured by flow cytometry for GFP expression following UBC-GFP-bGH-pA sequence knock-in at the CCR5 locus in T cells with varying concentrations of AZD7648 using RNP/AAV6 gene editing (n = 3). c. Percentage of viable cell count for the CCR5 gene targeted T cells (b) at 72 h post gene editing (n = 3) relative to the mock cells. d. Frequency of gene targeted cells as measured by flow cytometry for GFP following UBC-GFP-bGH-pA sequence knock-in at the CCR5 locus in T cells with or without AZD7648 (0.5 μM) treatment using a fixed amount of RNP and varying amounts of AAV6 donor (n = 3). e. Percentage of viable cell count for the CCR5 gene targeted T cells (d) at 72 h post gene editing (n = 3) relative to the mock cells. f. T cells were gene targeted at the TRAC locus for knock-in of CD19-CAR with and without AZD treatment (n = 3). Engineered CD19 CAR T cells were challenged with GFP+ Nalm6 leukemia target cells in co-culture at an effector to target ratio of 1:1 for 72 hours. Potency of the CAR T cell cytotoxicity activity was monitored by the residual percentage of GFP+ target cells by flow cytometry at 24 h and 48 h post challenge. All data are shown as mean ± SEM.

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Extended Data Fig. 10 AZD7648 improves gene targeting in B cells and HBECs.

a. Allelic distribution of mean WT, INDEL and HDR frequencies following gene editing at the CCR5 locus for knock-in of UBC-GFP-bGH-pA sequence in B cells with varying concentrations of AZD7648 using RNP/AAV6 gene editing (n = 2). HDR frequency was measured by ddPCR. WT and INDEL frequencies were measured through ICE analysis. Mean HDR to INDEL ratio is represented above the bars. b. Schematic for gene targeting at exon 1 of the CFTR gene for knock-in of SFFV-Citrine-pA sequence using RNP/AAV6 gene editing. c. Frequency of gene targeted cells as measured by flow cytometry for Citrine expression following CFTR-exon 1 gene editing (b) with or without AZD7648 treatment (0.5 μM) in HBECs (n = 4). d. Fold change in the frequency of CFTR gene targeted HBECs (c) with AZD7648 (0.5 μM) treatment relative to the untreated cells (n = 4). All data in c and d are shown as mean ± SEM.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–14.

Reporting Summary

Supplementary Table 1

This table includes gRNA sequences, antibodies, ddPCR primer/probe sequences and PCR primer sequences.

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Selvaraj, S., Feist, W.N., Viel, S. et al. High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01888-4

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