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; Martí Morera-Gómez; Andrea Barghetti; Sergi Muyo Abad; Roland F Baumgartner; Kedar N Natarajan; Lars K Nielsen; Tanya Warnecke; Ryan T Gill

doi:10.1016/j.isci.2023.108287

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

iScience. 2023 Oct 27;26(12):108287. doi: 10.1016/j.isci.2023.108287. eCollection 2023 Dec 15.

Authors

Arsenios Vlassis¹, Tanja L Jensen¹, Marina Mohr¹, Dominika J Jedrzejczyk¹, Xiangyou Meng¹, Gergo Kovacs², Martí Morera-Gómez¹, Andrea Barghetti^{1

3}, Sergi Muyo Abad¹, Roland F Baumgartner^{1

3}, Kedar N Natarajan², Lars K Nielsen^{1

4}, Tanya Warnecke^{1

3}, Ryan T Gill^{1

3}

Affiliations

¹ Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark.
² DTU Bioengineering, Technical University of Denmark, Søltofts Plads 224, 2800 Kongens Lyngby, Denmark.
³ Artisan Bio, 363 Centennial Parkway, Suite 310, Louisville, CO 80027, USA.
⁴ Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072, Australia.

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.

Keywords: Cell biology; Molecular Genetics; Molecular biology; Stem cells research; Techniques in genetics.