Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors

Authors:
Marta Holstein, Cristina Mesa-Nuñez, Csaba Miskey, Elena Almarza, Valentina Poletti, Marco Schmeer, Esther Grueso, Juan Carlos Ordóñez Flores, Dennis Kobelt, Wolfgang Walther, Manish K. Aneja, Johannes Geiger, Halvard B. Bonig, Zsuzsanna Izsvák, Martin Schleef, Carsten Rudolph, Fulvio Mavilio,Juan A. Bueren, Guillermo Guenechea, and Zoltán Ivics
In:
Source: Mol Ther
Publication Date: (2018)
Issue: 26(4): 1137-1153
Research Area:
Immunotherapy / Hematology
Stem Cells
Gene Expression
Regenerative medicine
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
Platform:
4D-Nucleofector™ X-Unit
Experiment

1 x 106 cells per sample were nucleofected with 10 µg SB transposon and 5 µg SB transposase construct, both provided either in plasmid DNA or MC DNA form. In a mass-to-mass comparison, equal amounts of both MC SB vectors in relation to their plasmid counterparts were tested; in an equimolar comparison, the amount of both MC SB vectors was adjusted in order to provide the same numbers of vector particles as in the plasmid sample. Since one of our objectives was to reduce DNA amount-dependent nucleofection toxicity, no compensation with filler DNA was applied. In some control experiments, cells were nucleofected with 10 µg SB transposon alone or
were subjected to nucleofection without DNA. In SNIM.RNA-based gene delivery experiments, the DNA vector carrying the SB100X transposase was replaced by 5 or 10 µg SNIM.RNA-SB. Cells were nucleofected using the 4D Nucleofector (Lonza) and E0100 program. After nucleofection, cells were resuspended in 2 mL complete
StemSpan medium.

Abstract

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.