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Chromatin opening elements allow tetracycline inducible gene expression in stem cells

In modern biomedicine, cell therapeutics are produced from reprogrammed stem cells (induced pluripotent stem cells, iPS). For this purpose, these cells can also undergo an additional genetic modification. However, unintentional mechanisms frequently occur, which can deactivate gene expression. Researchers from the Paul-Ehrlich-Institut (PEI) have developed inducible vectors containing ubiquitous chromatin opening elements (UCOE) which keep the relevant gene modified locus permanently open so that it remains active. The journal Biomaterials reports on these results in its online version of 24 November 2018.

25 / 2018

Ever since the discovery of the reprogramming of normal (somatic) body cells in so-called induced pluripotent stem cells (iPS cells), the development of cell therapeutics from such pluripotent stem cells has gathered momentum. While somatic cells represent specialized cells and are predetermined for one particular cell type, pluripotent stem cells can differentiate into quite a variety of different cell types. This very property is used for the development of cell therapeutics.

Not only the conversion of a normal body cell into the induced pluripotent stem cells is of scientific interest, but the road from these "formable" cells into the desired cell type (effector cell), too, represents a challenge in research. This can be supported, among other things, by transcription factors inserted by genetic modification of the pluripotent cells. Only the activity in defined time windows will generate a benefit. Insertion of the foreign genetic information into the cells is frequently carried out using retroviral vectors. Defective cells can thus be corrected, or the efficiency of somatic cell production from iPSC can be enhanced.

During this process, however, integrated retroviral vector genomes are often “silenced” by epigenetic mechanisms. This involves methyl groups, which are attached to certain gene sections (transgenic expression cassettes). These methyl groups have a kind of sealing effect. The advantage of these newly inserted genes is then lost, and even the differentiation of the cells can be stopped.

How can silencing be prevented without affecting inducibility? Professor Ute Modlich's research group "Gene Modification in Stem Cells", Division Veterinary Medicine of the PEI and colleagues of Division Medical Biotechnology developed inducible vectors which keep the gene modified site open by means of ubiquitous chromatin opening elements.

For this purpose, they inserted various fragments of the human HNRPA2B1-CBX3 UCOE into inducible retroviral vectors. The vectors can be induced using a promoter which is in turn activated by the antibiotic tetracycline. This one vector contains all that is necessary. Thanks to this property, it is also referred to as "all-in-one vector".

In a next step, the researchers tested the functionality of the vectors in various cell lines and in iPSCs of mice and men. They were able to prove that inserting the UCOE would not impair the regulation of the vectors. Switching them on – which is important for good therapeutic efficacy – and off was possible without any problems. This, in turn, is important to avoid effects of interference. Some of the tested vectors preserved their activity in iPSC of mice and humans, both in the pluripotency, and in the differentiation stage.

From the researchers’ point of view, these new vectors allow genetic modification of iPS cells with safe regulation of transgene expression. In a next step, the researchers swill use the vectors to efficiently produce blood cells from iPS cells.

Induced transgene expression (GFP, green fluorescence) in first stages of differentiation from iPS cells. Comparison between transmitted light and fluorescence microscopy.Induced transgene expression (GFP, green fluorescence) in first stages of differentiation from iPS cells. Comparison between transmitted light and fluorescence microscopy. Source: PEI

Original publication

Cullmann K, Blokland KEC, Sebe A, Schenk F, Ivics Z, Heinz N, Modlich U (2018): Sustained and regulated gene expression by Tet-inducible "All-In-One" retroviral vectors containing the HNRPA2B1-CBX3 UCOE.
Biomaterials Nov 24 [Epub ahead of print].
Online-Abstract

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The Paul-Ehrlich-Institut, the Federal Institute for Vaccines and Biomedicines, in Langen near Frankfurt/Main is a senior federal authority reporting to the Federal Ministry of Health (Bundesministerium für Gesundheit, BMG). It is responsible for the research, assessment, and marketing authorisation of biomedicines for human use and immunological veterinary medicinal products. Its remit also includes the authorisation of clinical trials and pharmacovigilance, i.e. recording and evaluation of potential adverse effects.

Other duties of the institute include official batch control, scientific advice and inspections. In-house experimental research in the field of biomedicines and life science form an indispensable basis for the manifold tasks performed at the institute.

The Paul-Ehrlich-Institut, with its roughly 800 members of staff, also has advisory functions nationally (federal government, federal states (Länder)), and internationally (World Health Organisation, European Medicines Agency, European Commission, Council of Europe etc.).

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