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Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair

Nature Biomedical Engineering (2023)Cite this article

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Abstract

The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1–6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.

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Fig. 1: Temporal optimization enhances delivery, expansion and targeted knock-in in human T cells.
Fig. 2: HMEJ-mediated integration allows for high-efficiency, targeted knock-in.
Fig. 3: Linearization of template is required for the effective integration of short homology HMEJ template.
Fig. 4: HMEJ allows for the efficient integration of very large templates.
Fig. 5: HMEJ-engineered CD19 CAR T cells are phenotypically similar to lentivirally transduced cells.
Fig. 6: CD19 CAR T cells control Raji tumours in vivo regardless of the engineering method.

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Data availability

The NGS sequencing data are available from the NCBI’s repository with the biosample accession numbers SAMN37790969, SAMN37790970, SAMN37790971, SAMN37790972, SAMN37790973, SAMN37790974, SAMN37790975, SAMN37790976, SAMN37790977 and SAMN37790978. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Garbie and the University of Minnesota Genomic Core for their assistance throughout the project. J.G.S. is supported by the T32HL007062-46 Hematology Research Training Program. B.S.M. is supported by the following NIH grants (CA136393, CA254849, AI161017, and AI161017). B.R.W. received support from NIH AI163731, CA237789, CA276345, the Minnesota Ovarian Cancer Alliance in 2021 and the Randy Shaver Cancer Research and Community Fund in 2022.

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Author notes

  1. These authors contributed equally: Beau R. Webber, Matthew J. Johnson.

Authors and Affiliations

  1. Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA

    Beau R. Webber, Matthew J. Johnson, Joseph G. Skeate, Nicholas J. Slipek, Walker S. Lahr, Anthony P. DeFeo, Lauren J. Mills, Xiaohong Qiu, Blaine Rathmann, Miechaleen D. Diers, Bryce Wick & Branden S. Moriarity

  2. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA

    Beau R. Webber, Matthew J. Johnson, Joseph G. Skeate, Nicholas J. Slipek, Walker S. Lahr, Anthony P. DeFeo, Lauren J. Mills, Xiaohong Qiu, Blaine Rathmann, Miechaleen D. Diers, Bryce Wick, Timothy K. Starr & Branden S. Moriarity

  3. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA

    Beau R. Webber, Matthew J. Johnson, Joseph G. Skeate, Nicholas J. Slipek, Walker S. Lahr, Anthony P. DeFeo, Xiaohong Qiu, Blaine Rathmann, Miechaleen D. Diers, Bryce Wick, R. Scott McIvor & Branden S. Moriarity

  4. Intima Bioscience, New York, NY, USA

    Tom Henley & Modassir Choudhry

  5. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA

    Timothy K. Starr & R. Scott McIvor

  6. Department of Ob-Gyn and Women’s Health, University of Minnesota, Minneapolis, MN, USA

    Timothy K. Starr

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Contributions

B.R.W., M.J.J., J.G.S., N.J.S., T.H., M.C., R.S.M. and B.S.M. conceived and planned the experiments. B.R.W., M.J.J., J.G.S., N.J.S., W.S.L., A.P.D., X.Q., B.R., M.D.D. and B.W. carried out the experiments. B.R.W., M.J.J., J.G.S., T.K.S., L.J.M. and B.S.M. analysed data and interpreted the results. B.R.W., M.J.J., J.G.S. and B.S.M. wrote the manuscript. All authors provided critical feedback during the study and commented on the manuscript.

Corresponding authors

Correspondence to Beau R. Webber or Branden S. Moriarity.

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Competing interests

B.R.W., R.S.M. and B.S.M. are principal investigators of Sponsored Research Agreements funded by Intima Biosciences to support this work. Patents have been filed covering the methods and approaches outlined in this work.

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Nature Biomedical Engineering thanks Zoltán Ivics, Zsuzsanna Izsvak, Matthew Porteus and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Longer homology arms increase integration with both HMEJ or HR.

Percentage of cells expressing GFP following electroporation with Cas9 mRNA, gRNAs, and HMEJ templates with the indicated homology arm lengths. Statistical analyses were done using Two-way ANOVA (n = 3–6 independent biological donors) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Source data

Extended Data Fig. 2 Lentivirally transduced cells have higher expression levels of CD19 CAR than non-viral engineered cells.

MFI of samples following HMEJ non-viral genome engineering or lentiviral transduction with a construct encoding CD19CAR-2A-RQR8.

Source data

Extended Data Fig. 3 HMEJ-engineered CD19 CAR T cells express similar levels of activation and exhaustion surface markers as lentivirally transduced cells.

(A) Expression of 41BB, CD25, CD69, and Ox40 in RQR8+CD4+, total CD4+, RQR8+CD8+ and total CD8+ T cell subsets following HMEJ non-viral genome engineering or lentiviral transduction with a construct encoding CD19CAR-2A-RQR8. (B) Expression of Lag3, PD1, and TIM3 in RQR8+CD4+, total CD4+, RQR8+CD8+ and total CD8+ T cell subsets following HMEJ non-viral genome engineering or lentiviral transduction with a construct encoding CD19CAR-2A-RQR8. Lack of statistically significant differences was determined by comparing TRAC, AAVS1, and pulse-only cells to Lenti cells using One-way ANOVA followed by Dunnett’s multiple comparison test.

Source data

Extended Data Fig. 4 HMEJ-engineered and lentivirally transduced CD19 CAR T cells produce cytokines in response to target cells, as measured by ICS.

Percentage of cells expressing cytokines IFNγ, TNF, and IL2 as well as a degranulation marker CD107a in CD4 (left panels) and CD8 (right panels) CD19 CAR T cells following coculture with CD19+ Raji target cells.

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Extended Data Fig. 5 HMEJ-engineered and lentivirally transduced CD19 CAR T cells produce cytokines in response to target cells, as measured by Luminex.

Concentration of IFNγ, TNF, IL4, and IL5 in the supernatant of CD19 CAR T cells following co-culture with CD19+ Raji target cells. All statistical analyses were done using One-way ANOVA followed by Tukey’s multiple comparison test. (n = 6 independent biological donors) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

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Webber, B.R., Johnson, M.J., Skeate, J.G. et al. Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01157-4

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