Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery.
Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse cells.
1] Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79108 Freiburg, Germany [2] Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany [3] Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany [4].
Sylwia
S
Bobis-Wozowicz
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
Melanie
M
Galla
1] Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79108 Freiburg, Germany [2] Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany [3] Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
Jamal
J
Alzubi
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
Johannes
J
Kuehle
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
Christopher
C
Baum
1] Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany [2] Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Axel
A
Schambach
1] Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79108 Freiburg, Germany [2] Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany [3] Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
Toni
T
Cathomen
D000818:Animals; D002460:Cell Line; D004720:Endonucleases / Q000235:genetics*; D055786:Gene Knockout Techniques; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D051379:Mice; D012190:Retroviridae / Q000235:genetics
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004720:Endonucleases