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AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

Nature Biomedical Engineering volume 8, pages 132–148 (2024)Cite this article

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Abstract

Engineering cells for adoptive therapy requires overcoming limitations in cell viability and, in the efficiency of transgene delivery, the duration of transgene expression and the stability of genomic integration. Here we report a gene-delivery system consisting of a Sleeping Beauty (SB) transposase encoded into a messenger RNA delivered by an adeno-associated virus (AAV) encoding an SB transposon that includes the desired transgene, for mediating the permanent integration of the transgene. Compared with lentiviral vectors and with the electroporation of plasmids of transposon DNA or minicircle DNA, the gene-delivery system, which we named MAJESTIC (for ‘mRNA AAV–SB joint engineering of stable therapeutic immune cells’), offers prolonged transgene expression, as well as higher transgene expression, therapeutic-cell yield and cell viability. MAJESTIC can deliver chimeric antigen receptors (CARs) into T cells (which we show lead to strong anti-tumour activity in vivo) and also transduce natural killer cells, myeloid cells and induced pluripotent stem cells with bi-specific CARs, kill-switch CARs and synthetic T-cell receptors.

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Fig. 1: Development of MAJESTIC, a composite mRNA:AAV-SB system for highly efficient generation of therapeutic immune cells.
Fig. 2: Comparison between MAJESTIC and conventional CAR T-cell generation approaches.
Fig. 3: Application of MAJESTIC for delivery of different therapeutic transgenes into T human cells.
Fig. 4: Comparison of MC transposon and MAJESTIC methods.
Fig. 5: Genomic insertion and safe harbour preference of MC transposon and MAJESTIC methods.
Fig. 6: CAR-NK generation via MAJESTIC.
Fig. 7: CAR-monocyte and CAR-MA generation via MAJESTIC.

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Data availability

The main data supporting the results in this study are available within the paper and its supplementary information. The raw and processed genome-sequence data from the splinkerette experiments are available from the NIH Sequence Read Archive/Gene Expression Omnibus under the accession number GSE220202 (token mtqrayyonvwfzmd). The raw and analysed datasets generated during the study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

The scripts used to process the insertion site-mapping data are available at https://github.com/stanleyzlam/SB-CAR.

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Acknowledgements

We thank all members of the Chen Laboratory and of various entities at the University of Yale for discussions. We thank various Yale core facilities for technical support. In particular, we thank K. Tang and P. Renauer for technical assistance on Illumina sequencing and data analysis. We also thank C. Miskey for sharing processed sequence files from ref. 54. S.C. is supported by a Yale SBI/Genetics Startup Fund and by grants from NIH/NCI/NIDA (DP2CA238295, R01CA231112, R33CA225498, RF1DA048811), DoD (W81XWH-20-1-0072, W81XWH-21-10514), Alliance for Cancer Gene Therapy, Sontag Foundation (DSA), Pershing Square Sohn Cancer Research Alliance, Yale Cancer Center Pilot Award, Dexter Lu Gift, Ludwig Family Foundation, and Chenevert Family Foundation. S.Z.L. is supported by Yale College Fellowships.

Author information

Author notes

  1. Lupeng Ye

    Present address: Institute of Modern Biology, Nanjing University, Nanjing, China

Authors and Affiliations

  1. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

    Lupeng Ye, Stanley Z. Lam, Luojia Yang, Kazushi Suzuki, Yongji Zou, Qianqian Lin, Yueqi Zhang, Paul Clark, Lei Peng & Sidi Chen

  2. System Biology Institute, Yale University, West Haven, CT, USA

    Lupeng Ye, Stanley Z. Lam, Luojia Yang, Kazushi Suzuki, Yongji Zou, Qianqian Lin, Yueqi Zhang, Paul Clark, Lei Peng & Sidi Chen

  3. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA

    Lupeng Ye, Stanley Z. Lam, Luojia Yang, Kazushi Suzuki, Yongji Zou, Qianqian Lin, Yueqi Zhang, Paul Clark, Lei Peng & Sidi Chen

  4. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA

    Luojia Yang & Sidi Chen

  5. Immunobiology Program, Yale University, New Haven, CT, USA

    Sidi Chen

  6. Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  7. Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  8. Yale Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

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Contributions

S.C. conceived the study. L. Ye designed the experiments with assistance from S.Z.L. L. Ye performed most experiments, with assistance from S.Z.L., L.P., K.S., Q.L., Y.Z. and P.C. L. Yang, K.S. and Y.Z. performed revision experiments with technical supervision by L.P. S.Z.L. and K.S. analysed next-generation sequencing data. L. Ye, S.Z.L., L. Yang and S.C. prepared the paper, with inputs from all authors. S.C. secured funding and supervised the work.

Corresponding author

Correspondence to Sidi Chen.

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Competing interests

A patent related to this study was filed by Yale University (inventors: S.C., L.Y. and S.Z.L.) and licensed to Cellinfinity Bio, a Yale biotech start-up founded by S.C. S.C. is also a (co)founder of EvolveImmune Tx, Chen Consulting, Chen Tech and NumericGlobal, all unrelated to this study. The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Harjeet Singh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 MAJESTIC CAR generation and optimization.

a, Schematic of AAV-SB-CD22.CAR, AAV-SB-BCMA.CAR, and SB100X constructs and key procedures: mRNA in vitro transcription, AAV production, mRNA electroporation, flow cytometry, and kill assay. b, Representative flow cytometry plots of AAV-SB-BCMA.CAR CD4 (left) and CD8 (right) T cells show the percentage of CAR-expressing cells. CD4 cells are defined as CD3+ and CD8− cells. c, Quantification of CD22.CAR T cell ratio of (Fig. 1b, c). d, Quantification of BCMA.CAR T ratio in human CD4 and CD8 T cells. In this figure, for optimization of conditions, each assay was done with one donor with three technical replicates. Donor 2 T cells were used in this figure.

Source data

Extended Data Fig. 2 Timepoint optimization, SB100X transposase mRNA titration, and MAJESTIC CAR T yield.

a, Quantification of CD22.CAR T cell ratio for CD4 T cells that were transduced with AAV-SB-CD22.CAR virus at various time points. b, Quantification of CD22.CAR T cell ratio for CD8 T cells that were transduced with AAV-SB-CD22.CAR virus at various time points. c-d, Representative flow cytometry plots of CD22.CAR T cells produced via AAV-SB and a titrated serial of SB100X mRNA. (c) CD8 T cells. (d) CD4 T cells. e, Quantification of the cell viability of CD8 T cells. f, Quantification of CAR T cells. g, Flow cytometry plots of CAR T cell ratios before and after sorting. h, CD22.CAR T cell yield quantification (yield = total viable cell count x CAR-positive percentage). Cells were split into 3 technical replicates after electroporation. Yield is calculated for each technical replicate separately. i, CAR+ T cell generation efficiency (CAR+%) of MAJESTIC using Neon and Maxcyte approaches. In this figure, for optimization of conditions, each assay was done with one donor with three technical replicates. Donor 2 and donor 0286 T cells were used in this figure.

Source data

Extended Data Fig. 3 Vector-copy-number quantification, immune-marker profiling, and functionality testing of MAJESTIC-produced CD8 CAR T cells.

a-c, Vector copy number (VCN) quantification of MAJESTIC-manufactured CAR-T cells. Purified CAR T cells were collected for DNA extraction after three weeks of mRNA electroporation and viral transduction. (a) left arm probe, (b) right arm probe, (c) left panel: left arm probe, right panel: right arm probe. d, SB100X transposase excision efficiency evaluation. Left panel: left arm probe, right panel: right arm probe. e, Cytolysis analysis of NAML6-GL (NAML6 with GFP and luciferase reporters) cancer cells that were co-cultured with Lenti-CD22.CAR and AAV-SB-CD22.CAR T cells. CAR-Ts were seeded at various effector:target (E:T) ratios, and luciferase imaging was performed at two time points (16h and 40h). f, Cytolysis analysis of MM.1R-GL (MM.1R with GFP and luciferase reporters) cancer cells that were co-cultured with Lenti-BCMA.CAR and AAV-SB-BCMA.CAR T cells. CAR-Ts were seeded at various effector : target (E:T) ratios, and luciferase imaging was performed at two time points (16h and 40h). g, Exhaustion and memory marker expression in CD22-CAR and HER2-CAR T cells before and post transfection. Unpaired t tests were performed to evaluate statistical significance. h, Bioluminescent density of NSG mice that were injected with NALM6-GL cancer cells and with CD22-CAR therapy (n = 7 mice per group). i, Quantification of total luminescence for (h). n = 7 mice. Two-way ANOVA with multiple comparisons tests was performed to evaluate statistical significance. j, Survival curve of NALM6-GL-induced leukemia-bearing NSG mice that treated with PBS, untreated CD8 T cells, and AAV-SB-CD22.CAR T cells. Log-rank (Mantel-Cox) tests were performed to evaluate statistical significance. Donor 0007, 4003, 5003, 003C, 0286 T cells were used in this figure. Significance notes: ns - not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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Extended Data Fig. 4 Generation of various therapeutic immune cells by MAJESTIC.

a, Quantification of the HER2.CAR T cell viability. b, Quantification of HER2.CAR-positive CD8 T cells. c, Representative flow-cytometry plots of EGFRvIII.CAR-NK92 cells were produced via either the SB system or lentiviral transduction. d, Quantification of (c). e, Quantification of CD19.20.CAR T cells. f, Flow cytometry detection of CD19 and CD20 expression in NALM6-GL cells. g, Cytolysis analysis of NALM6-GL cancer cells that were co-cultured with lenti-CD19.20.CAR and AAV-SB-CD19.20.CAR T cells. Donor 2 and donor VP2 T cells were used in this figure.

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Extended Data Fig. 5 Generation of various therapeutic immune cells by MAJESTIC.

a, Representative flow cytometry plots of NY-ESO-1 T cells. b, Quantification of (a). c, Quantification of the CD22.CAR.iCasp T cell viability. d, Representative flow cytometry plots of CD22.CAR.iCasp9 T cells. e, Quantification of (d). f, Quantification of the CD22.CAR.iCasp9 T cells post antigen-specific cancer cells stimulation. In this figure, each assay has three technical replicates, donor 601c and donor 02 T cells were used in this figure.

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Extended Data Fig. 6 Gene-delivery efficiency comparison of MC-SB/SB100X mRNA with the MAJESTIC system.

a, Flow-cytometry histogram overlays and bar plots of cell viability post-electroporation as measured with 7-AAD staining. b, Flow-cytometry data of CD22.CAR T cells from human primary CD3 T cells produced by plasmid transposon plasmid, transposon MC, transposon MC with mRNA-transposase, and MAJESTIC. c, Quantification of (b). d, Yield calculations (yield = CAR% * total viable cell count). All conditions started with an equal amount of primary T cells per replicate. Three CAR% replicates were averaged and then multiplied by the average of 2 cell count replicates. Left panel, total T cell count; Right panel, total CAR+ T cell count. e, Quantification of flow-cytometry data of CD22.CAR T cells from four human PBMCs (same data as Fig. 4e–f, plotted in dot-whisker plots).

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Supplementary information

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Supplementary Fig. 1 and Table 1.

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Supplementary Data 1

List of key oligos.

Supplementary Data 2

Statistical data for Supplementary Fig. 1.

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Ye, L., Lam, S.Z., Yang, L. et al. AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells. Nat. Biomed. Eng 8, 132–148 (2024). https://doi.org/10.1038/s41551-023-01058-6

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