SEED-Selection enables high-efficiency enrichment of primary T cells edited at multiple loci

Christopher R Chang; Vivasvan S Vykunta; Jae Hyun J Lee; Ke Li; Clara Kochendoerfer; Joseph J Muldoon; Charlotte H Wang; Thomas Mazumder; Yang Sun; Daniel B Goodman; William A Nyberg; Chang Liu; Vincent Allain; Allison Rothrock; Chun J Ye; Alexander Marson; Brian R Shy; Justin Eyquem

doi:10.1038/s41587-024-02531-6

SEED-Selection enables high-efficiency enrichment of primary T cells edited at multiple loci

Nat Biotechnol. 2025 Dec;43(12):2043-2053. doi: 10.1038/s41587-024-02531-6. Epub 2025 Feb 5.

Authors

Christopher R Chang^{1

2

3

4}, Vivasvan S Vykunta^{1

2

3

4}, Jae Hyun J Lee^{1

5}, Ke Li^{1

5}, Clara Kochendoerfer^{1

2

4

6}, Joseph J Muldoon^{1

2}, Charlotte H Wang^{1

2

4}, Thomas Mazumder⁷, Yang Sun^{2

7}, Daniel B Goodman^{1

2

6

8}, William A Nyberg^{1

2}, Chang Liu^{1

2}, Vincent Allain^{1

2

9}, Allison Rothrock^{1

2}, Chun J Ye^{1

2

7

10

11

12

13

14

15

16}, Alexander Marson^{1

2

6

10

11

17

18}, Brian R Shy^{19

20

21}, Justin Eyquem^{22

23

24

25

26

27

28}

Affiliations

¹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
² Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
³ Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.
⁴ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁵ Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA.
⁶ Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA.
⁷ Division of Rheumatology, University of California, San Francisco, San Francisco, CA, USA.
⁸ Diabetes Center, University of California, San Francisco, San Francisco, CA, USA.
⁹ Université Paris Cité, INSERM UMR976, Hôpital Saint-Louis, Paris, France.
¹⁰ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
¹¹ Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA.
¹² Bakar Computational Health Sciences Institute, University of California, San Francisco, San Francisco, CA, USA.
¹³ Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, CA, USA.
¹⁴ Arc Institute, Palo Alto, CA, USA.
¹⁵ Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA.
¹⁶ Bakar Aging Research Institute, University of California, San Francisco, San Francisco, CA, USA.
¹⁷ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
¹⁸ UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
¹⁹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. Brian.Shy@ucsf.edu.
²⁰ Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA. Brian.Shy@ucsf.edu.
²¹ UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. Brian.Shy@ucsf.edu.
²² Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. Justin.Eyquem@ucsf.edu.
²³ Department of Medicine, University of California, San Francisco, San Francisco, CA, USA. Justin.Eyquem@ucsf.edu.
²⁴ Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA. Justin.Eyquem@ucsf.edu.
²⁵ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. Justin.Eyquem@ucsf.edu.
²⁶ Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. Justin.Eyquem@ucsf.edu.
²⁷ Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. Justin.Eyquem@ucsf.edu.
²⁸ UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. Justin.Eyquem@ucsf.edu.

Abstract

Engineering T cell specificity and function at multiple loci can generate more effective cellular therapies, but current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here we develop a one-step process to enrich unlabeled cells containing knock-ins at multiple target loci using a family of repair templates named synthetic exon expression disruptors (SEEDs). SEEDs associate transgene integration with the disruption of a paired target endogenous surface protein while preserving target expression in nonmodified and partially edited cells to enable their removal (SEED-Selection). We design SEEDs to modify three critical loci encoding T cell specificity, coreceptor expression and major histocompatibility complex expression. The results demonstrate up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). This method is compatible with existing clinical manufacturing workflows and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.

MeSH terms

Exons / genetics
Gene Editing* / methods
Gene Knock-In Techniques
Genetic Loci
Humans
T-Lymphocytes* / cytology
T-Lymphocytes* / metabolism

Abstract

MeSH terms

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