/ / / / A highly efficient transgene knock-in technology in clinically relevant cell types

A highly efficient transgene knock-in technology in clinically relevant cell types

, ,
featured image

Data availability

Source data are provided with this paper. Unprocessed flow cytometry and microscopy images for Figs. 1–5 are provided as Source Data File 1, and numerical values for plotted data in Figs. 1–5 are provided as Source Data File 2. DNA constructs, gRNAs, primers, ddPCR probes, Digenome-Seq results and rhAmp-Seq panel of potential off-target candidate sites are listed in the Supplementary Table. All numerical data values used to generate figures in the Supplementary Information can be found in Supplementary Data. Raw flow cytometry plots and representative gating strategies, as well as uncropped microscopy and gel images, are provided in the Supplementary Information under Supplementary Notes. High-throughput sequencing data have been deposited in the National Center for Biotechnology Informationʼs Sequence Read Archive database (accession code PRJNA947757) and can be found at http://www.ncbi.nlm.nih.gov/bioproject/947757 (ref. 62). Source data are provided with this paper.

Code availability

Custom code used to analyze Digenome-Seq data is available at the Editas Medicine GitHub page at https://github.com/editasmedicine/digenomitas. Custom code used to identify candidate in silico off-target sites is also available at the Editas Medicine GitHub (https://github.com/editasmedicine/calitas).

References

  1. Bailey, S. R. & Maus, M. V. Gene editing for immune cell therapies. Nat. Biotechnol. 37, 1425–1434 (2019).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  2. Raffin, C., Vo, L. T. & Bluestone, J. A. Treg cell-based therapies: challenges and perspectives. Nat. Rev. Immunol. 20, 158–172 (2020).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  3. Wilkinson, A. C., Igarashi, K. J. & Nakauchi, H. Haematopoietic stem cell self-renewal in vivo and ex vivo. Nat. Rev. Genet. 21, 541–554 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  4. Raje, N. et al. Anti-BCMA CAR T-cell therapy bb2121 in relapsed or refractory multiple myeloma. N. Engl. J. Med. 380, 1726–1737 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  5. Abramson, J. S. Anti-CD19 CAR T-cell therapy for B-cell non-Hodgkin lymphoma. Transfus. Med. Rev. 34, 29–33 (2020).

    Article 
    PubMed 

    Google Scholar 

  6. Frigault, M. J. et al. Tisagenlecleucel CAR T-cell therapy in secondary CNS lymphoma. Blood 134, 860–866 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  7. Naeimi Kararoudi, M. et al. Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV. Cell Rep. Methods 2, 100236 (2022).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  8. Xie, G. et al. CAR-NK cells: a promising cellular immunotherapy for cancer. EBioMedicine 59, 102975 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  9. Liu, E. et al. Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. N. Engl. J. Med. 382, 545–553 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  10. Depil, S., Duchateau, P., Grupp, S. A., Mufti, G. & Poirot, L. ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat. Rev. Drug. Discov. 19, 185–199 (2020).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  11. Srpan, K. et al. Shedding of CD16 disassembles the NK cell immune synapse and boosts serial engagement of target cells. J. Cell Biol. 217, 3267–3283 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  12. Snyder, K. M. et al. Expression of a recombinant high affinity IgG Fc receptor by engineered NK cells as a docking platform for therapeutic mAbs to target cancer cells. Front. Immunol. 9, 2873 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  13. Hu, W., Wang, G., Huang, D., Sui, M. & Xu, Y. Cancer immunotherapy based on natural killer cells: current progress and new opportunities. Front. Immunol. 10, 1205 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  14. Szmania, S. et al. Ex vivo-expanded natural killer cells demonstrate robust proliferation in vivo in high-risk relapsed multiple myeloma patients. J. Immunother. 38, 24–36 (2015).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  15. Giron-Michel, J. et al. Membrane-bound and soluble IL-15/IL-15Rα complexes display differential signaling and functions on human hematopoietic progenitors. Blood 106, 2302–2310 (2005).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  16. Weber, E. W., Maus, M. V. & Mackall, C. L. The emerging landscape of immune cell therapies. Cell 181, 46–62 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  17. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  18. 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).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  19. Zhang, L. et al. AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines. Nat. Commun. 12, 3908 (2021).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  20. Bulcha, J. T., Wang, Y., Ma, H., Tai, P. W. L. & Gao, G. Viral vector platforms within the gene therapy landscape. Signal Transduct. Target. Ther. 6, 53 (2021).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  21. Jasin, M. & Rothstein, R. Repair of strand breaks by homologous recombination. Cold Spring Harb. Perspect. Biol. 5, a012740 (2013).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  22. Kao, T. et al. GAPTrap: a simple expression system for pluripotent stem cells and their derivatives. Stem Cell Rep. 7, 518–526 (2016).

    Article 
    CAS 

    Google Scholar 

  23. Liu, Z. et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci. Rep. 7, 2193 (2017).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  24. Dobosy, J. R. et al. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers. BMC Biotechnol. 11, 80 (2011).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  25. Park, J. B., Park, H., Son, J., Ha, S. J. & Cho, H. S. Structural study of monomethyl fumarate-bound human GAPDH. Mol. Cells 42, 597–603 (2019).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  26. Yilmaz, A., Peretz, M., Aharony, A., Sagi, I. & Benvenisty, N. Defining essential genes for human pluripotent stem cells by CRISPR–Cas9 screening in haploid cells. Nat. Cell Biol. 20, 610–619 (2018).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  27. Eisenberg, E. & Levanon, E. Y. Human housekeeping genes, revisited. Trends Genet. 29, 569–574 (2013).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  28. Laoharawee, K. et al. Genome engineering of primary human B cells using CRISPR/Cas9. J. Vis. Exp. 10.3791/61855 (2020).

  29. Pomeroy, E. J. et al. A genetically engineered primary human natural killer cell platform for cancer immunotherapy. Mol. Ther. 28, 52–63 (2020).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  30. Robert, F., Barbeau, M., Ethier, S., Dostie, J. & Pelletier, J. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med. 7, 93 (2015).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  31. Roth, T. L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405–409 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  32. Nguyen, D. N. et al. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nat. Biotechnol. 38, 44–49 (2020).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  33. Oh, S. A. et al. High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA. J. Exp. Med. 219, e20211530 (2022).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  34. Shy, B. R. et al. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat. Biotechnol. (2022).

  35. Fennell, T. et al. CALITAS: a CRISPR–Cas-aware ALigner for In silico off-TArget Search. CRISPR J. 4, 264–274 (2021).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  36. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  37. Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  38. Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  39. Bothmer, A. et al. Detection and modulation of DNA translocations during multi-gene genome editing in T cells. CRISPR J. 3, 177–187 (2020).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  40. Ivancic, D. et al. INSERT-seq enables high-resolution mapping of genomically integrated DNA using Nanopore sequencing. Genome Biol. 23, 227 (2022).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  41. Wang, Y., Zhao, Y., Bollas, A., Wang, Y. & Au, K. F. Nanopore sequencing technology, bioinformatics and applications. Nat. Biotechnol. 39, 1348–1365 (2021).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  42. Sondka, Z. et al. The COSMIC Cancer Gene Census: describing genetic dysfunction across all human cancers. Nat. Rev. Cancer 18, 696–705 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  43. Pickup, M., Novitskiy, S. & Moses, H. L. The roles of TGFβ in the tumour microenvironment. Nat. Rev. Cancer 13, 788–799 (2013).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  44. Gornalusse, G. G. et al. HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nat. Biotechnol. 35, 765–772 (2017).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  45. Zhang, L. et al. Author Correction: AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines. Nat. Commun. 12, 4500 (2021).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  46. Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nat. Genet. 53, 895–905 (2021).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  47. Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  48. Keppel, M. P., Saucier, N., Mah, A. Y., Vogel, T. P. & Cooper, M. A. Activation-specific metabolic requirements for NK cell IFN-γ production. J. Immunol. 194, 1954–1962 (2015).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  49. Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science 367, eaba7365 (2020).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  50. Ho, J. Y. et al. Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo. Mol. Ther. Methods Clin. Dev. 21, 237–246 (2021).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  51. Cichocki, F. et al. iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti-PD-1 therapy. Sci. Transl. Med. 12, eaaz5618 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  52. Kath, J. et al. Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells. Mol. Ther. Methods Clin. Dev. 25, 311–330 (2022).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  53. Ng, E. S., Davis, R. P., Azzola, L., Stanley, E. G. & Elefanty, A. G. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood 106, 1601–1603 (2005).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  54. Decker, T. & Lohmann-Matthes, M. L. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Methods 115, 61–69 (1988).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  55. Allicotti, G., Borras, E. & Pinilla, C. A time-resolved fluorescence immunoassay (DELFIA) increases the sensitivity of antigen-driven cytokine detection. J. Immunoassay Immunochem. 24, 345–358 (2003).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  56. Uhlen, M. et al. Proteomics. Tissue-based map of the human proteome. Science 347, 1260419 (2015).

    Article 
    PubMed 

    Google Scholar 

  57. Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 25, 229–233 (2019).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  58. Zhang, J., Kobert, K., Flouri, T. & Stamatakis, A. PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 30, 614–620 (2014).

    Article 
    CAS 
    PubMed 

    Google Scholar 

  59. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  60. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  61. Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  62. Marco, E. A highly efficient transgene knock-in technology in clinically relevant cell types. National Center for Biotechnology Information. http://www.ncbi.nlm.nih.gov/bioproject/947757 (2023).

Download references

Acknowledgements

We would like to thank additional members of the Editas Computational Biology, Informatics and Sequencing groups for generation and pipeline analysis of sequencing data. We thank R. Naines, C. Wang, J. Yao and H. An for providing primary cells for studies. We would like to thank J. Getgano, K. Gareau, E. Goncz, S. Zhang, J. Moon, K. Tsiounis and J. Schafer for support in the development of key assays and protocols. We would like to thank members of BlueRock Therapeutics LP for their support and collaboration related to engineering and culturing iPSCs. We would like to thank A. Dee for paper preparation support. Several graphics in the figures (cells in Fig. 1a, cells in Fig. 2a,h, cells in Fig. 3a,d,g, mouse schematic in Fig. 5a, cells in Supplementary Fig. 5c, cells in Supplementary Fig. 9c,e, cells in Supplementary Fig. 10a, cells in Supplementary Fig. 11a–c, cells in Supplementary Fig. 12c, cells in Supplementary Fig. 13a and cells in Supplementary Fig. 14a,b) were created with BioRender. We would like to thank Porterhouse Medical for graphic design support.

Author information

Author notes

  1. These authors contributed equally: Alexander G. Allen, Samia Q. Khan, Carrie M. Margulies, Ramya Viswanathan.

Authors and Affiliations

  1. Editas Medicine, Cambridge, MA, USA

    Alexander G. Allen, Samia Q. Khan, Carrie M. Margulies, Ramya Viswanathan, Swarali Lele, Laura Blaha, Sean N. Scott, Kaitlyn M. Izzo, Alexandra Gerew, Rithu Pattali, Nadire R. Cochran, Carl S. Holland, Amy H. Zhao, Stephen E. Sherman, Michael C. Jaskolka, Meng Wu, Aaron C. Wilson, Xiaoqi Sun, Dawn M. Ciulla, Deric Zhang, Jacqueline D. Nelson, Peisheng Zhang, Patrizia Mazzucato, Yan Huang, Georgia Giannoukos, Eugenio Marco, Michael Nehil, John A. Follit, Kai-Hsin Chang, Mark S. Shearman, Christopher J. Wilson & John A. Zuris

Contributions

A.G.A, S.Q.K., C.M.M., R.V., S.L., S.N.S., K.M.I., A.G., R.P., N.R.C., C.S.H., A.H.Z., S.E.S., M.C.J., M.W., A.C.W., D.M.C., D.Z., Y.H., J.D.N., P.Z. and P.M. performed experiments and analyzed data. L.B., X.S., J.A.F. and J.A.Z. analyzed data. A.G.A., S.Q.K., C.M.M., R.V., S.L., L.B., S.E.S., M.C.J., X.S., G.G., E.M., M.N., J.A.F., K.Z., K.-H.C., M.S.S., C.J.W. and J.A.Z. wrote the paper.

Corresponding author

Correspondence to
John A. Zuris.

Ethics declarations

Competing interests

All authors were employees and shareholders of Editas Medicine at the time the work was performed. J.A.Z. and C.M.M. are inventors on patent WO2021226151A2 that has been filed by Editas Medicine relating to this work.

Peer review

Peer review information

Nature Biotechnology thanks Fyodor Urnov and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–18 as well as raw images of supplementary figure flow cytometry plots, microscopy images and western blot uncropped gels.

Reporting Summary

Supplementary Table 1

List of plasmid constructs, gRNAs, ddPCR and RT–qPCR primers, Digenome-Seq and rHampSeq specificity data.

Supplementary Data 1

Numerical data used to generate graphed figures in the Supplementary Information file.

Source data

About this article

Verify currency and authenticity via CrossMark

Cite this article

Allen, A.G., Khan, S.Q., Margulies, C.M. et al. A highly efficient transgene knock-in technology in clinically relevant cell types.
Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01779-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41587-023-01779-8

Read More

Share on Google Plus
    Blogger Comment
    Facebook Comment

0 Comments :

Post a Comment

Subscribe to: Post Comments ( Atom )