TY  - JOUR
A1  - Holstein, Marta
A1  - Mesa-Nuñez, Cristina
A1  - Miskey, Csaba
A1  - Almarza, Elena
A1  - Poletti, Valentina
A1  - Schmeer, Marco
A1  - Grueso, Esther
A1  - Ordóñez Flores, Juan Carlos
A1  - Kobelt, Dennis
A1  - Walther, Wolfgang
A1  - Aneja, Manish Kumar
A1  - Geiger, Johannes
A1  - Bönig, Halvard-Björn
A1  - Izsvák, Zsuzsanna
A1  - Schleef, Martin
A1  - Rudolph, Carsten
A1  - Mavilio, Fulvio
A1  - Bueren, Juan
A1  - Guenechea, Guillermo
A1  - Ivics, Zoltán
T1  - Efficient non-viral gene delivery into human hematopoietic stem cells by minicircle sleeping beauty transposon vectors
T2  - Molecular therapy
N2  - The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4–8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.
KW  - chromosomal integration
KW  - gene therapy
KW  - gene vectors
KW  - hematopoietic stem cells
KW  - nonviral gene delivery
KW  - transposition
Y1  - 2018
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/50129
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-501298
SN  - 1525-0024
SN  - 1525-0016
N1  - This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
VL  - 26
IS  - 4
SP  - 1137
EP  - 1153
PB  - Elsevier ; Nature Publ. Group
CY  - Amsterdam ; New York, NY
ER  -