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Efficient non-viral immune cell engineering using circular single-stranded DNA-mediated genomic integration

Nature Biotechnology volume 43, pages 1821–1832 (2025)Cite this article

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Abstract

The use of adeno-associated viruses (AAVs) as donors for homology-directed repair (HDR)-mediated genome engineering is limited by safety issues, manufacturing constraints and restricted packaging limits. Non-viral targeted genetic knock-ins rely primarily on double-stranded DNA (dsDNA) and linear single-stranded DNA (lssDNA) donors. dsDNA is known to have low efficiency and high cytotoxicity, while lssDNA is challenging for scaled manufacture. In this study, we developed a non-viral genome writing catalyst (GATALYST) system that allows production of circular single-stranded DNAs (cssDNAs) up to approximately 20 kilobases as donor templates for highly efficient precision transgene integration. cssDNA donors enable knock-in efficiency of up to 70% in induced pluripotent stem cells (iPSCs) and improved efficiency in multiple clinically relevant primary immune cell types and at multiple genomic loci implicated for clinical applications with various nuclease editor systems. The high precision and efficiency in chimeric antigen receptor (CAR)-T and natural killer (NK) cells, improved safety, payload flexibility and scalable manufacturability of cssDNA shows potential for future applications of genome engineering.

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Fig. 1: Scalable cssDNA manufacturing and purification from bacteriophage.
Fig. 2: GFP reporter knock-in efficiency and cell viability in K562 cells.
Fig. 3: iPSC engineering with cssDNA.
Fig. 4: Human primary T cell engineering with cssDNA.
Fig. 5: cssDNA non-viral genomic engineering for human primary NK cells, B cells and CD34+ HSPCs.
Fig. 6: Efficient functional CAR-T cell engineering with cssDNA.

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Data availability

RNA-seq data have been submitted to the Gene Expression Omnibus under accession code GSE278608 (ref. 51).

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Acknowledgements

We thank T. Cha for designing and cloning initial phagemid constructs and for guidance and suggestions on cssDNA production. We thank Quintara Biosciences for plasmid construction and DNA/RNA sequencing service for this project; LumiGenics for the in vivo CAR-T functional assays; and MaxCyte for technical support and suggestions for the electroporation experiment. We also thank M. C. Lorence for comments and suggestions on the manuscript. Cartoon illustrations were generated with BioRender. This study was partially funded by Small Business Innovation Research (SBIR) to Stellate DNA LLC (grant award ID: 2052290).

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Authors and Affiliations

  1. Full Circles Therapeutics, Cambridge, MA, USA

    Keqiang Xie, Jakob Starzyk, Ishita Majumdar, Jiao Wang, Katerina Rincones, Thao Tran, Danna Lee, Sarah Niemi, John Famiglietti, Richard Shan & Hao Wu

  2. Stellate DNA, Cambridge, MA, USA

    Bernhard Suter, Richard Shan & Hao Wu

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  1. Keqiang Xie
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Contributions

H.W., R.S. and K.X. conceived the idea for this project. K.X. and H.W. designed the experiments and interpreted the data. K.X., J.S., I.M., J.W., K.R. T.T., D.L., S.N. and J.F. performed the experiments. K.X. and H.W. oversaw the study. K.X. and H.W. wrote the manuscript, with input from all other authors.

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Correspondence to Keqiang Xie or Hao Wu.

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Competing interests

K.X., J.S., I.M., J.W., K.R., T.T., D.L., S.N., J.F., R.S. and H.W. are either current or former employees of Full Circles Therapeutics. Patents related to this study have been filed. The other authors declare no competing interests.

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Xie, K., Starzyk, J., Majumdar, I. et al. Efficient non-viral immune cell engineering using circular single-stranded DNA-mediated genomic integration. Nat Biotechnol 43, 1821–1832 (2025). https://doi.org/10.1038/s41587-024-02504-9

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