CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells

Arsenios Vlassis, Tanja L. Jensen, Marina Mohr, Dominika J. Jedrzejczyk, Xiangyou Meng, Gergo Kovacs, Andrea Barghetti, Martí Morera-Gómez, Sergi Muyo Abad, Roland F. Baumgartner, Kedar N. Natarajan, Lars K. Nielsen, Tanya Warnecke, Ryan T. Gill*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

Discovery of genomic safe harbor sites (SHSs) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CARs), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in induced pluripotent stem cells (iPSCs), primary T and natural killer (NK) cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC toward CD34+ hematopoietic stem and progenitor cells (HSPCs), lymphoid progenitor cells (LPCs), and NK cells and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHSs can be used to engineer cells for adoptive immunotherapies.
Original languageEnglish
Article number108287
JournaliScience
Volume26
Issue number12
Number of pages30
ISSN2589-0042
DOIs
Publication statusPublished - 2023

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