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Original article
Bacteria-free minicircle DNA system to generate integration-free CAR-T cells
  1. Chen Cheng1,2,
  2. Na Tang2,
  3. Jiaxin Li3,
  4. Shiwei Cao2,4,
  5. Tongtong Zhang2,4,
  6. Xiaofei Wei5,
  7. Haoyi Wang2,4
  1. 1 School of Life Sciences, University of Science and Technology of China, Hefei, China
  2. 2 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China
  3. 3 Northwest Agriculture and Forestry University, Yangling, China
  4. 4 University of the Chinese Academy of Sciences, Beijing, China
  5. 5 Beijing Cord Blood Bank, Beijing, China
  1. Correspondence to Dr Haoyi Wang, University of the Chinese Academy of Sciences, Beijing 101408, China; wanghaoyi{at}


Background Chimeric antigen receptor T (CAR-T) cells engineered with lentiviral and retroviral vectors have been successfully applied to treat patients with B cell malignancy. However, viral integration in T cells has the potential risk of mutagenesis, and viral vector production demands effort and is costly. Using non-integrative episomal vector such as minicircle vector to generate integration-free CAR-T cells is an attractive option.

Methods and results We established a novel method to generate minicircle vector within a few hours using simple molecular biology techniques. Since no bacteria is involved, we named these vectors bacteria-free (BF) minicircle. In comparison with plasmids, BF minicircle vector enabled higher transgene expression and improved cell viability in human cell line, stem cells and primary T cells. Using BF minicircle vector, we generated integration-free CAR-T cells, which eliminated cancer cells efficiently both in vitro and in vivo.

Conclusion BF minicircle vector will be useful in basic research as well as in clinical applications such as CAR-T and gene therapy. Although the transgene expression of minicircle vector lasts apparently shorter than that of insertional lentivirus, multiple rounds of BF minicircle CAR-T cell infusion could eliminate cancer cells efficiently. On the other hand, a relatively shorter CAR-T cell persistence provides an opportunity to avoid serious side effects such as cytokine storm or on-target off-tumour toxicity.

  • bacteria-free minicircle vector
  • integration free car-t cells
  • cell viability
  • human Cd34+ Hscs
  • human es cells

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  • CC and NT contributed equally.

  • Contributors CC, NT and HW designed the experiments. CC, NT, JL, SC, TZ and XW performed the experiments. CC, NT and HW wrote the manuscript. All authors discussed and interpreted the results.

  • Funding This study was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no: XDA01010409, XDA16010205), State 863 project (grant no: 2015AA020307), National Natural Science Foundation of China (10.13039/501100001809) (grant nos: 31471215 and 81773269), and the National Key Research and Development Program of China (no 2016YFA0101402). HW is supported by the ’Young Thousand Talent Project'.

  • Competing interests None declared.

  • Patient consent Obtained.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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