Evaluation of TCR Gene Editing Achieved by TALENs, CRISPR/Cas9, and megaTAL Nucleases.
Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies.
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.|Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA.|Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA.
Mark J
MJ
Osborn
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Beau R
BR
Webber
German Cancer Research Center (DKFZ), Heidelberg, Germany.|Department of Translational Oncology, National Center for Tumor Diseases, Heidelberg, Germany.
Friederike
F
Knipping
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Cara-lin
CL
Lonetree
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Nicole
N
Tennis
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Anthony P
AP
DeFeo
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Amber N
AN
McElroy
Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA.|Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, Washington, USA.
Colby G
CG
Starker
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Catherine
C
Lee
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Sarah
S
Merkel
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Troy C
TC
Lund
Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, Washington, USA.
Karen S
KS
Kelly-Spratt
Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, Washington, USA.|Department of Pediatrics, University of Washington, Seattle, Washington, USA.
Michael C
MC
Jensen
Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA.|Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA.
Daniel F
DF
Voytas
German Cancer Research Center (DKFZ), Heidelberg, Germany.|Department of Translational Oncology, National Center for Tumor Diseases, Heidelberg, Germany.
Christof
C
von Kalle
German Cancer Research Center (DKFZ), Heidelberg, Germany.|Department of Translational Oncology, National Center for Tumor Diseases, Heidelberg, Germany.
Manfred
M
Schmidt
German Cancer Research Center (DKFZ), Heidelberg, Germany.|Department of Translational Oncology, National Center for Tumor Diseases, Heidelberg, Germany.
Richard
R
Gabriel
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Keli L
KL
Hippen
Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA.
Jeffrey S
JS
Miller
Seattle Children's Research Institute, and University of Washington School of Medicine, Seattle, Washington, USA.
Andrew M
AM
Scharenberg
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.|Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA.
Jakub
J
Tolar
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Bruce R
BR
Blazar
D001665:Binding Sites; D064113:CRISPR-Cas Systems; D018929:Cell Culture Techniques; D002460:Cell Line; D004720:Endonucleases; D000072669:Gene Editing; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D056426:Genetic Loci; D016678:Genome; D006801:Humans; D016130:Immunophenotyping; D009369:Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D013601:T-Lymphocytes / Q000378:metabolism; D000069896:Transcription Activator-Like Effector Nucleases; D014161:Transduction, Genetic
D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D004720:Endonucleases; D000069896:Transcription Activator-Like Effector Nucleases