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Nature communications
01 02, 2024;15 (1): 111.

Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells.

Ron Baik, M Kyle Cromer, Steve E Glenn, Christopher A Vakulskas, Kay O Chmielewski, Amanda M Dudek, William N Feist, Julia Klermund, Suzette Shipp, Toni Cathomen, Daniel P Dever, Matthew H Porteus
Author Information
  1. Ron Baik: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive, Stanford, CA, USA. ORCID
  2. M Kyle Cromer: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive, Stanford, CA, USA. ORCID
  3. Steve E Glenn: Integrated DNA Technologies, Inc., Coralville, IA, USA.
  4. Christopher A Vakulskas: Integrated DNA Technologies, Inc., Coralville, IA, USA. ORCID
  5. Kay O Chmielewski: Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106, Freiburg, Germany.
  6. Amanda M Dudek: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive, Stanford, CA, USA.
  7. William N Feist: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive, Stanford, CA, USA. ORCID
  8. Julia Klermund: Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106, Freiburg, Germany. ORCID
  9. Suzette Shipp: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive, Stanford, CA, USA.
  10. Toni Cathomen: Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106, Freiburg, Germany. ORCID
  11. Daniel P Dever: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive, Stanford, CA, USA.
  12. Matthew H Porteus: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive, Stanford, CA, USA. mporteus@stanford.edu. ORCID
PMID: 38169468 DOI: 10.1038/s41467-023-43413-w

Abstract

Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach to increasing the frequency of HDR in human HSPCs by the delivery of an inhibitor of 53BP1 (named "i53") as a recombinant peptide. We show that the use of i53 peptide effectively increases the frequency of HDR-mediated genome editing at a variety of therapeutically relevant loci in HSPCs as well as other primary human cell types. We show that incorporating the use of i53 recombinant protein allows high frequencies of HDR while lowering the amounts of AAV6 needed by 8-fold. HDR edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in NSG mice, suggesting that i53 recombinant protein might be safely integrated into the standard CRISPR/AAV6-mediated genome editing protocol to gain greater numbers of edited cells for transplantation of clinically meaningful cell populations.

References

Porteus, M. H. A new class of medicines through DNA editing. N. Engl. J. Med. 380, 947–959 (2019). [PMID: 30855744]
Yeh, C. D., Richardson, C. D. & Corn, J. E. Advances in genome editing through control of DNA repair pathways. Nat. Cell Biol. 21, 1468–1478 (2019). [PMID: 31792376]
Frangoul, H. et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N. Engl. J. Med. 384, 252–260 (2021). [PMID: 33283989]
Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216–1224 (2018). [PMID: 30082871]
Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). [PMID: 27820943]
Bak, R. O., Dever, D. P. & Porteus, M. H. CRISPR/Cas9 genome editing in human hematopoietic stem cells. Nat. Protoc. 13, 358–376 (2018). [PMID: 29370156]
Bak, R. O. et al. Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6. Elife 6, e27873 (2017).
Charlesworth, C. T. et al. Priming human repopulating hematopoietic stem and progenitor cells for cas9/sgrna gene targeting. Mol. Ther. Nucleic Acids 12, 89–104 (2018). [PMID: 30195800]
Pavel-Dinu, M. et al. Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nat. Commun. 10, 1634 (2019). [PMID: 30967552]
Martin, R. M. et al. Improving the safety of human pluripotent stem cell therapies using genome-edited orthogonal safeguards. Nat. Commun. 11, 2713 (2020). [PMID: 32483127]
Vaidyanathan, S. et al. High-efficiency, selection-free gene repair in airway stem cells from cystic fibrosis patients rescues CFTR function in differentiated epithelia. Cell Stem Cell 26, 161–171.e4 (2020). [PMID: 31839569]
DeWitt, M. A. et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci. Transl. Med. 8, 360ra134 (2016). [PMID: 27733558]
Genovese, P. et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature 510, 235–240 (2014). [PMID: 24870228]
Wang, J. et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat. Biotechnol. 33, 1256–1263 (2015). [PMID: 26551060]
Schiroli, G. et al. Precise gene editing preserves hematopoietic stem cell function following transient p53-mediated DNA damage response. Cell Stem Cell 24, 551–565.e8 (2019). [PMID: 30905619]
Hirsch, M. L. et al. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells. PLoS One 6, e27520 (2011). [PMID: 22114676]
Adams, M. M. & Carpenter, P. B. Tying the loose ends together in DNA double strand break repair with 53BP1. Cell Div. 1, 19 (2006). [PMID: 16945145]
Yuan, J. & Chen, J. N terminus of CtIP is critical for homologous recombination-mediated double-strand break repair. J. Biol. Chem. 284, 31746–31752 (2009). [PMID: 19759395]
Escribano-Díaz, C. et al. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Mol. Cell 49, 872–883 (2013). [PMID: 23333306]
Canny, M. D. et al. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat. Biotechnol. 36, 95–102 (2018). [PMID: 29176614]
Nambiar, T. S. et al. Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant. Nat. Commun. 10, 3395 (2019). [PMID: 31363085]
Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013). [PMID: 23258413]
Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013). [PMID: 23258412]
Cromer, M. K. et al. Global transcriptional response to CRISPR/Cas9-AAV6-based genome editing in CD34+ hematopoietic stem and progenitor cells. Mol. Ther. 26, 2431–2442 (2018). [PMID: 30005866]
Skryabin, B. V. et al. Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9-mediated genome editing events. Sci. Adv. 6, eaax2941 (2020). [PMID: 32095517]
Norris, A. L. et al. Template plasmid integration in germline genome-edited cattle. Nat. Biotechnol. 38, 163–164 (2020). [PMID: 32034391]
De Ravin, S. S. et al. Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells. Blood 137, 2598–2608 (2021). [PMID: 33623984]
Ward, I. M., Minn, K., van Deursen, J. & Chen, J. p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice. Mol. Cell. Biol. 23, 2556–2563 (2003). [PMID: 12640136]
Liu, X. et al. Overlapping functions between XLF repair protein and 53BP1 DNA damage response factor in end joining and lymphocyte development. Proc. Natl. Acad. Sci. USA 109, 3903–3908 (2012). [PMID: 22355127]
Piras, F. et al. Lentiviral vectors escape innate sensing but trigger p53 in human hematopoietic stem and progenitor cells. EMBO Mol. Med. 9, 1198–1211 (2017). [PMID: 28667090]
Georgakilas, A. G., Martin, O. A. & Bonner, W. M. p21: a two-faced genome guardian. Trends Mol. Med. 23, 310–319 (2017). [PMID: 28279624]
Lattanzi, A. et al. Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease. Sci. Transl. Med. 13, eabf2444 (2021).
Damian, M. & Porteus, M. H. A crisper look at genome editing: RNA-guided genome modification. Mol. Ther. 21, 720–722 (2013). [PMID: 23542565]
Cromer, M. K. et al. Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells. Nat. Med. 27, 677–687 (2021). [PMID: 33737751]
Gomez-Ospina, N. et al. Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I. Nat. Commun. 10, 4045 (2019). [PMID: 31492863]
Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015). [PMID: 26121415]
Wiebking, V. et al. Genome editing of donor-derived T-cells to generate allogenic chimeric antigen receptor-modified T cells: optimizing αβ T cell-depleted haploidentical hematopoietic stem cell transplantation. Haematologica 106, 847–858 (2021). [PMID: 32241852]
Srifa, W. et al. Cas9-AAV6-engineered human mesenchymal stromal cells improved cutaneous wound healing in diabetic mice. Nat. Commun. 11, 2470 (2020). [PMID: 32424320]
Taheri-Ghahfarokhi, A. et al. Decoding non-random mutational signatures at Cas9 targeted sites. Nucleic Acids Res. 46, 8417–8434 (2018). [PMID: 30032200]
Bae, S. & Kim, J.-S. Machine learning finds Cas9-edited genotypes. Nat. Biomed. Eng. 2, 892–893 (2018). [PMID: 31015728]
Nakauchi, Y. et al. The cell type-specific 5hmC landscape and dynamics of healthy human hematopoiesis and TET2-mutant preleukemia. Blood Cancer Discov. 3, 346–367 (2022). [PMID: 35532363]
Turchiano, G. et al. Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq. Cell Stem Cell 28, 1136–1147.e5 (2021). [PMID: 33626327]
Wienert, B., Wyman, S. K., Yeh, C. D., Conklin, B. R. & Corn, J. E. CRISPR off-target detection with DISCOVER-seq. Nat. Protoc. 15, 1775–1799 (2020). [PMID: 32313254]
Sharma, R. et al. The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells. Nat. Commun. 12, 472 (2021).
Ferrari, S. et al. Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells. Cell Stem Cell 29, 1428–1444.e9 (2022). [PMID: 36206730]
Ferrari, S. et al. Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking. Nat. Biotechnol. 38, 1298–1308 (2020). [PMID: 32601433]
Lehnertz, B. et al. HLF expression defines the human hematopoietic stem cell state. Blood 138, 2642–2654 (2021). [PMID: 34499717]
Brault, J. et al. CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells. Front. Immunol. 13, 1067417 (2022). [PMID: 36685559]
Tao, J., Bauer, D. E. & Chiarle, R. Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing. Nat. Commun. 14, 212 (2023). [PMID: 36639728]
Hanlon, K. S., Kleinstiver, B. P., Garcia, S. P. & Zaborowski, M. P. High levels of AAV vector integration into CRISPR-induced DNA breaks. Nat Commun. 10, 4439 (2019). [PMID: 31570731]
Mantri, S. et al. The Binns Program for Cord Blood Research: a novel model of cord blood banking for academic biomedical research. Placenta 103, 50–52 (2021). [PMID: 33075720]
Wiebking, V. et al. Metabolic engineering generates a transgene-free safety switch for cell therapy. Nat. Biotechnol. 38, 1441–1450 (2020). [PMID: 32661439]
Aurnhammer, C. et al. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum. Gene Ther. Methods 23, 18–28 (2012). [PMID: 22428977]
Rhiel, M. et al. T-CAST: an optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds. Front. Genome Ed. 5, 1130736 (2023). [PMID: 36890979]

Grants

  1. P30 CA008748/NCI NIH HHS
  2. R01 HL135607/NHLBI NIH HHS

MeSH Term

Humans
Animals
Mice
Gene Editing
Hematopoietic Stem Cells
Hematopoietic Stem Cell Transplantation
Recombinant Proteins
Peptides
CRISPR-Cas Systems

Chemicals

Recombinant Proteins
Peptides
Journal Article Research Support, N.I.H., Intramural Research Support, Non-U.S. Gov't

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