Efficient non-viral gene delivery into human hematopoietic stem cells by minicircle sleeping beauty transposon vectors

  • 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.
Author:Marta Holstein, Cristina Mesa-Nuñez, Csaba Miskey, Elena Almarza, Valentina Poletti, Marco Schmeer, Esther Grueso, Juan Carlos Ordóñez Flores, Dennis Kobelt, Wolfgang Walther, Manish Kumar Aneja, Johannes Geiger, Halvard-Björn BönigORCiDGND, Zsuzsanna Izsvák, Martin Schleef, Carsten Rudolph, Fulvio Mavilio, Juan Bueren, Guillermo Guenechea, Zoltán Ivics
Pubmed Id:https://pubmed.ncbi.nlm.nih.gov/29503198
Parent Title (English):Molecular therapy
Publisher:Elsevier ; Nature Publ. Group
Place of publication:Amsterdam ; New York, NY
Document Type:Article
Year of Completion:2018
Date of first Publication:2018/01/31
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2019/04/29
Tag:chromosomal integration; gene therapy; gene vectors; hematopoietic stem cells; nonviral gene delivery; transposition
Page Number:17
First Page:1137
Last Page:1153
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Institutes:Medizin / Medizin
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - Namensnennung-Nicht kommerziell - Keine Bearbeitung 4.0