Cas9-Cleavage Sequences in Size-Reduced Plasmids Enhance Nonviral Genome Targeting of CARs in Primary Human T Cells

Ruirui Jing; Peng Jiao; Jiangqing Chen; Xianhui Meng; Xiaoyan Wu; Yanting Duan; Kai Shang; Liling Qian; Yanjie Huang; Junwei Liu; Tao Huang; Jin Jin; Wei Chen; Xun Zeng; Weiwei Yin; Xiaofei Gao; Chun Zhou; Michel Sadelain; Jie Sun

doi:10.1002/smtd.202100071

Cas9-Cleavage Sequences in Size-Reduced Plasmids Enhance Nonviral Genome Targeting of CARs in Primary Human T Cells

Small Methods. 2021 Jul;5(7):e2100071. doi: 10.1002/smtd.202100071. Epub 2021 May 19.

Authors

Ruirui Jing^{1

2

3}, Peng Jiao^{1

2

3}, Jiangqing Chen^{1

2

3}, Xianhui Meng^{1

2

3}, Xiaoyan Wu^{1

2

3}, Yanting Duan^{1

2

3}, Kai Shang^{1

2

3}, Liling Qian^{1

2}, Yanjie Huang⁴, Junwei Liu⁵, Tao Huang⁶, Jin Jin⁶, Wei Chen⁷, Xun Zeng⁸, Weiwei Yin⁵, Xiaofei Gao⁴, Chun Zhou⁹, Michel Sadelain¹⁰, Jie Sun^{1

2

3}

Affiliations

¹ Department of Cell Biology and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.
² Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China.
³ Zhejiang Laboratory for Systems & Precision Medicine, Zhejiang University Medical Center, Hangzhou, 310058, China.
⁴ Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, 310058, China.
⁵ Key Laboratory for Biomedical Engineering of the Ministry of Education, College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou, 310058, China.
⁶ MOE Laboratory of Biosystem Homeostasis and Protection and Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China.
⁷ Department of Neurobiology, Institute of Neuroscience, and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.
⁸ State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, and First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.
⁹ School of Public Health, and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.
¹⁰ Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, New York, NY, 10044, USA.

PMID: 34927998
DOI: 10.1002/smtd.202100071

Abstract

T cell genome editing holds great promise to advance a range of immunotherapies but is encumbered by the dependence on difficult-to-produce and expensive viral vectors. Here, small double-stranded plasmid DNA modified to mediate high-efficiency homologous recombination is designed. The resulting chimeric antigen receptor (CAR)-T cells display a similar phenotype, transcriptional profile, and in vivo potency to CAR-T cells generated using adeno-associated viral vector. This method should simplify and accelerate the use of precision engineering to produce edited T cells for research and clinical purposes.

Keywords: CAR; CRISPR/Cas9; adoptive cell therapy; genome targeting; nonviral methods; size-reduced plasmid.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems*
DNA
Gene Editing / methods
Gene Targeting
Genetic Vectors
Homologous Recombination
Humans
Immunotherapy / methods
Immunotherapy, Adoptive / methods*
Mice
NIH 3T3 Cells
Plasmids*
T-Lymphocytes / immunology*

Substances

DNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding