Virus- and Stem cell biology

Virus and Stem Cell Biology

Section leader

Prof. Dr. Hoeben, R.C. (Rob)

Subgroup leaders

Prof. Dr. Hoeben, R.C. (Rob)
Dr. Mikkers, H.M.M. (Harald)
Dr. Gonçalves, M.A.F.V. (Manuel)

Other principal investigators

Dr. Zaldumbide, A (Arnaud)
Dr. Knaän-Shanzer, S. (Shosh)

Research overview

The research in the section of Virus & Stem Cell Biology focuses on the fundamental aspects of viruses and stem cells, and their development into therapeutic agents for the treatment of human inherited and acquired diseases.  A hallmark of our research is the combinatory use of cell and gene transfer technologies. Pre-clinical/clinical testing of these newly developed technologies is performed in collaboration with research groups within and outside the LUMC.

The virus biology research involves the development and characterization of improved viral vectors for biomedical research, in particular gene therapy. Three topics have our special interest:

"oncolytic viruses": This project is about developing viruses as anti-cancer agents. Our research is restricted to adenoviruses and reoviruses, which are two relatively harmless viruses. One approach is to modify the tropism of human adenoviruses, most of which normally cause only mild respiratory or gastrointestinal symptoms, by inclusion of tumor-specific ligands in their capsids. In parallel, we generate human reoviruses that selectively kill tumor cells. Our oncolytic viruses have been evaluated in models of prostate cancer, glioblastoma, liver cancer, and melanoma in collaboration with scientists in the Netherlands, France, Sweden, and the United Kingdom.

"immune evasion": To reduce the immune response of recipients after the transplantation of allogeneic cells or cells carrying specific transgenes we exploit several immune evasion strategies normally used by viruses. We have evaluated a cis-acting inhibitor of proteasomal degradation derived from the Epstein-Barr virus nuclear antigen-1. Moreover, we identified a new inhibitor peptide in the latency-associated nuclear antigen-1 of Kaposi’s sarcoma-associated herpesvirus. Both inhibitors prevented antigenic peptide generation from transgene products allowing the continuous expression of antigenic proteins in cells because they are not destructed by T-lymphocytes. In addition, we are currently investigating a number of other immune evasion strategies, such as expression of herpesvirus immunoevasins to render transplanted cells non-immunogenic. The focus of this work is to protect transplanted human insulin-producing beta cells from auto-and allo-reactive T cells.

“third-generation adenovirus vectors”: This research focuses on the development of improved production systems for gutless adenovirus vectors and their use for the efficient introduction of large or multiple transgenes into human progenitor and stem cells with minimal vector-related toxicity. In addition to the use of regular third-generation adenovirus vectors for the transient genetic modification of target cells, we have embarked on the generation of new vector types for stable transgene expression in transduced target cells using locus/site-specific transgene integration or homologous recombination. For example, we have generated new adenovirus/adeno-associated virus vectors. These vectors stably integrate into a specific locus on human chromosome 19 and are capable of genetically complementing dystrophin-deficient human myoblasts.

Our stem cell research involves various stem cell types including human hematopoietic and mesenchymal stem cells, and pluripotent stem cells, such as embryonic and induced pluripotent stem cells.

“directed stem cell differentiation”: This project aims at converting easily obtainable human cells like fibroblasts and mesenchymal stem cells into (progenitors of) skeletal muscle cells, cardiomyocytes, and insulin-producing beta-cells by forced expression of myogenic, cardiac, and pancreatic transcription factors, respectively. Such cells would be beneficial for fundamental (differentiation mechanisms) and applied (cell therapy) research. Furthermore, we study various culture conditions/growth factors to improve the differentiation of human mesenchymal stem cells into adipocytes, cardiomyocytes, chrondrocytes, myoblasts, osteoblasts, and pancreatic islet cells.

“non-coding RNAs and differentiation”: The genome contains as many coding as non-coding RNAs. We have specifically selected long non-coding RNAs that may be important for the maintenance of pluripotent stem cells on the one hand and differentiation (neural) on the other hand. We have genetically modified the endogenous loci containing the selected non-coding RNA genes in mouse embryonic stem cells to study their role in self-renewal and differentiation.

“induced pluripotent stem cells”: Induced pluripotent stem (iPS) cells are pluripotent cells resembling embryonic stem cells, which can be generated from differentiated somatic cells through forced expression of pluripotency-inducing factors. Our line of iPS cell research concerns both fundamental and translational aspects. We are interested in developing new episomal gene delivery techniques to efficiently derive human iPS cells which are better suited for clinical purposes. To study disease pathogenesis and the potential of iPS cells for cell therapies we are in the process of generating iPS cells from healthy individuals and individuals with specific inherited diseases. Furthermore, we are developing systems facilitating the analysis of the mechanisms underlying the reversal of somatic cells into pluripotent stem cells.

“tissue regeneration”: We are investigating the regenerative capacity of various human stem cell types in immunocompromised animals with experimentally or genetically-induced damage in the tibialis anterior muscle, heart, pancreas, or skin. To facilitate detection of the donor cells after transplantation, reporter genes are transferred into these cells.

In addition to the virus and stem cell biology research our section operates the LUMC viral vector facility. This facility produces viral vectors for preclinical research within the LUMC. The facility also handles requests for the production of viral vectors from our lentivirus vector-based short-hairpin expression library for knocking down the expression of ± 17.500 human and 16.000 mouse genes..

Recent highlights.

• Development of new methods for presenting ligands at the surface of the adenovirus capsid (J Virol 2004;78;3470-3479; J Virol 2005;79:3206-3210; Gene Ther 2007;14:664-670; Gene Ther 2008;15:978–9890.
• Development of reovirus platform for viral oncolysis (Gene Ther 2008;15:1567–1578; Clin. Cancer Res. 2011 17:2767-2776).
• Development of methods to inhibit presentation of antigens in the context of major histocompatibility complex type I molecules (Cancer Gene Ther 2006;13:584-591; Mol Immunol 2007;44;1352-1360; Mol Immunol 2007;44:3588-3596; Biotechnol. Lett. 2010 32: 749-754).
• Development of directed stem cell fusion as a new concept to genetically modify human skeletal muscle cells (Mol Ther 2008;16:741-748).
• Demonstrating the ability of hybrid viral vectors to mediate (locus-specific) integration of (large fragments of) foreign DNA into host cell chromosomes and to accomplish long-term transgene expression in rapidly proliferating cells (Virology 2004;321:287-296; J Virol 2005;79:3146-3162; PLoS ONE 2008;3:e3084).
• Generation and characterization of induced pluripotent stem cells (Exp Cell Res 2008;314: 3255-3263).
• Exploitation of our know-how in diabetes research (Islets 2010; 2: 164-173; Genes Immun. 2011; 12: 415-427; J. Immunol. 185(3):1412-1418)
• Use of our technology for ground breaking research (Nat Methods 2008;5:189-196; Nat Genet 2008;40:1354-1359).
• Participant in PERSIST, an FP7 Large-scale Integrating Project that will explore the use of innovative gene modification and delivery technologies for the treatment of monogenetic diseases.

Recent key publications

• Carlotti, F., Zaldumbide, A., Loomans, C.J., Van Rossenberg, E., Engelse, M., De Koning, E.J., Hoeben, R.C. (2010)  Isolated human islets contain a distinct population of mesenchymal stem cells. Islets 2: 164-173.
• Chadt, A., Leicht, K., Deshmukh, A., Jiang, L.Q., Scherneck, S., Bernhardt, U., Dreja, T., Vogel, H., Schmolz, K., Kluge, R., Zierath, J.R., Hultschig, C., Hoeben, R.C., Schürmann, A., Joost, H.G., Al-Hasani, H. (2008) A mutation in the positional candidate gene Tbc1d1 in the SJL mouse strain confers leanness and protects from high-fat diet induced obesity. Nat. Genet. 40:1354-1359.
• de la Garza-Rodea, A.S., van der Velde, I., Boersma, H., Gonçalves, M.A., van Bekkum, D.W., de Vries, A.A., Knaän-Shanzer, S. (2011) Long-term contribution of human bone marrow mesenchymal stromal cells to skeletal muscle regeneration in mice. Cell Transplant. 20:217-231.
• Duinsbergen, D, Eriksson, M., 't Hoen, P.A., Frisén, J., Mikkers, H.(2008) Induced pluripotency with endogenous and inducible genes. Exp Cell Res. 314:3255-63.
• Gonçalves MA, Holkers M, van Nierop GP, Wieringa R, Pau MG, de Vries AA. (2008) Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system. PLoS One. 3:e3084.
• Uil, T.G., Vellinga, J., De Vrij,J., Van den Hengel, S.K., Rabelink, M.J.W.E., Cramer, S.J., Eekels, J.J.M., Ariyurek, Y., van Galen, M., Hoeben, R.C. (2011) Directed adenovirus evolution using engineered mutator viral polymerases. Nucl. Acids Res. 39: e30.
• Uren, A.G., Mikkers, H., Koo,l J., van der Weyden, L., Lund, A.H., Wilson, C.H., Rance, R., Jonkers, J., van Lohuizen, M., Berns, A., Adams, D.J. (2009) A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites. Nat Protoc. 4:789-98.
• van Nierop, G.P., de Vries, A.A., Holkers, M., Vrijsen, K.R., Gonçalves, M.A. (2009)  Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease. Nucleic Acids Res. 2009; 37:5725-36.
• Wang, X., Herr, R.A., Rabelink, M., Hoeben, R.C., Wiertz, E.J., Hansen, TH. (2009) Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degradation substrates. J. Cell Biol.187: 655–668.
• Zaldumbide, A., Ossevoort, M., Wiertz, E.J.H.J., Hoeben, R.C. (2007) In-cis inhibition of antigen processing by the latency-associated nuclear antigen I of kaposi sarcoma herpes virus. Mol. Immunol. 44: 1352-1360.

Relation with other LUMC research themes

The combined expertise and the stem cell and viral vector technology is used internally for fundamental research projects, but also finds employment via the thematic group ‘Regenerative Medicine’ in more clinically oriented projects for various disease targets. Productive collaborations are in place with the department of Anatomy and Embryology and with several clinical research groups within the LUMC, as well as in national and international research consortia. Research performed in this group is well positioned in view of the plans to further strengthen stem cell research in the LUMC.

Keywords

• Adenovirus
• Adeno-associated virus
• Animal model
• Cancer
• Cardiovascular disease
• Diabetes
• Differentiation
• Embryonic stem cell
• Gene therapy
• Gene targeting
• Induced pluripotent stem cell
• Lentivirus
• Mesenchymal stem cell
• Muscular dystrophy
• Neural stem cell
• Reovirus
• Regenerative medicine
• Reprogramming
• Transcription factor
• Viral vector