Original Article

Gene Therapy (2017) 24, 298–307; doi:10.1038/gt.2017.20; published online 20 April 2017

Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells

D Hoffmann1,2,3, J W Schott1,2, F K Geis1,2, L Lange1,2, F-J Müller4, D Lenz1, D Zychlinski1, D Steinemann5, M Morgan1, T Moritz1,2 and A Schambach1,2,3,6

  1. 1Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
  2. 2Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany
  3. 3Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany
  4. 4Zentrum für Integrative Psychiatrie, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
  5. 5Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany
  6. 6Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

Correspondence: Professor A Schambach, Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail: schambach.axel@mh-hannover.de

Received 5 October 2016; Revised 27 January 2017; Accepted 6 March 2017
Advance online publication 20 April 2017

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Abstract

Correction of patient-specific induced pluripotent stem cells (iPSC) upon gene delivery through retroviral vectors offers new treatment perspectives for monogenetic diseases. Gene-modified iPSC clones can be screened for safe integration sites and differentiated into transplantable cells of interest. However, the current bottleneck is epigenetic vector silencing. In order to identify the most suitable retroviral expression system in iPSC, we systematically compared vectors from different retroviral genera, different promoters and their combination with ubiquitous chromatin opening elements (UCOE), and several envelope pseudotypes. Lentiviral vectors (LV) pseudotyped with vesicular stomatitis virus glycoprotein were superior to gammaretroviral and alpharetroviral vectors and other envelopes tested. The elongation factor 1α short (EFS) promoter mediated the most robust expression, whereas expression levels were lower from the potent but more silencing-prone spleen focus forming virus (SFFV) promoter. Both full-length (A2UCOE) and minimal (CBX3) UCOE juxtaposed to two physiological and one viral promoter reduced transgene silencing with equal efficiency. However, a promoter-specific decline in expression levels was not entirely prevented. Upon differentiation of transgene-positive iPSC into endothelial cells, A2UCOE.EFS and CBX3.EFS vectors maintained highest transgene expression in a larger fraction of cells as compared with all other constructs tested here. The function of UCOE diminished, but did not fully counteract, vector silencing and possibilities for improvements remain. Nevertheless, the CBX3.EFS in a LV background exhibited the most promising promoter and vector configuration for both high titer production and long-term genetic modification of human iPSC and their progeny.