TY - JOUR
T1 - CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells
AU - Vlassis, Arsenios
AU - Jensen, Tanja L.
AU - Mohr, Marina
AU - Jedrzejczyk, Dominika J.
AU - Meng, Xiangyou
AU - Kovacs, Gergo
AU - Barghetti, Andrea
AU - Morera-Gómez, Martí
AU - Muyo Abad, Sergi
AU - Baumgartner, Roland F.
AU - Natarajan, Kedar N.
AU - Nielsen, Lars K.
AU - Warnecke, Tanya
AU - Gill, Ryan T.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
U2 - 10.1016/j.isci.2023.108287
DO - 10.1016/j.isci.2023.108287
M3 - Journal article
C2 - 38034357
SN - 2589-0042
VL - 26
JO - iScience
JF - iScience
IS - 12
M1 - 108287
ER -