Virus and Stem Cell Biology
|Prof. Dr. Hoeben, R.C. (Rob)|
|Dr. Mikkers, H.M.M. (Harald)|
|Dr. Gonçalves, M.A.F.V. (Manuel)|
|Dr. Ressing, M (Maaike)|
|Dr. Zaldumbide, A (Arnaud)|
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 pancreatic cancer.
"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, and together with the department of Anatomy and Embryology, the LUMC iPS core 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. Moreover, a lentivirus-based library of 40.000 CRISPR genes targeting the human genes is available.
Recent key publications
Chen X, Rinsma M, Janssen JM, Liu J, Maggio I, Goncalves MA. Probing the impact of chromatin conformation on genome editing tools. Nucleic Acids Res 2016 Jul 27;44(13):6482-6492.
Dautzenberg IJ, van den Wollenberg DJ, van den Hengel SK, Limpens RW, Barcena M, Koster AJ, Hoeben RC. Mammalian orthoreovirus T3D infects U-118 MG cell spheroids independent of junction adhesion molecule-A. Gene Ther 2014 Jun;21(6):609-617.
de Jong VM, van der Slik AR, Laban S, van 't SR, Koeleman BP, Zaldumbide A, Roep BO. Survival of autoreactive T lymphocytes by microRNA-mediated regulation of apoptosis through TRAIL and Fas in type 1 diabetes. Genes Immun 2016 Sep;17(6):342-348.
Gram AM, Oosenbrug T, Lindenbergh MF, Bull C, Comvalius A, Dickson KJ, Wiegant J, Vrolijk H, Lebbink RJ, Wolterbeek R, Adema GJ, Griffioen M, Heemskerk MH, Tscharke DC, Hutt-Fletcher LM, Wiertz EJ, Hoeben RC, Ressing ME. The Epstein-Barr Virus Glycoprotein gp150 Forms an Immune-Evasive Glycan Shield at the Surface of Infected Cells. PLoS Pathog 2016 Apr;12(4):e1005550.
Hoelen H, Zaldumbide A, van Leeuwen WF, Torfs EC, Engelse MA, Hassan C, Lebbink RJ, de Koning EJ, Resssing ME, de Ru AH, van Veelen PA, Hoeben RC, Roep BO, Wiertz EJ. Proteasomal Degradation of Proinsulin Requires Derlin-2, HRD1 and p97. PLoS One 2015; 10(6):e0128206.
Holkers M, Maggio I, Henriques SF, Janssen JM, Cathomen T, Goncalves MA. Adenoviral vector DNA for accurate genome editing with engineered nucleases. Nat Methods 2014 Oct;11 (10):1051-1057.
Hoyng SA, Gnavi S, de WF, Eggers R, Ozawa T, Zaldumbide A, Hoeben RC, Malessy MJ, Verhaagen J. Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve. Gene Ther 2014 Jun;21 (6):549-557.
Maggio I, Liu J, Janssen JM, Chen X, Goncalves MA. Adenoviral vectors encoding CRISPR/Cas9 multiplexes rescue dystrophin synthesis in unselected populations of DMD muscle cells. Sci Rep 2016 Nov 15;6 :37051.
Maggio I, Stefanucci L, Janssen JM, Liu J, Chen X, Mouly V, Goncalves MA. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations. Nucleic Acids Res 2016 Feb 18;44 (3):1449-1470.
Mikkers HM, Freund C, Mummery CL, Hoeben RC. Cell replacement therapies: is it time to reprogram? Hum Gene Ther 2014 Oct;25(10):866-874.
van de Weijer ML, Bassik MC, Luteijn RD, Voorburg CM, Lohuis MA, Kremmer E, Hoeben RC, LeProust EM, Chen S, Hoelen H, Ressing ME, Patena W, Weissman JS, McManus MT, Wiertz EJ, Lebbink RJ. A high-coverage shRNA screen identifies TMEM129 as an E3 ligase involved in ER-associated protein degradation. Nat Commun 2014 May 8;5:3832.
van den Wollenberg DJ, Dautzenberg IJ, Ros W, Lipinska AD, van den Hengel SK, Hoeben RC. Replicating reoviruses with a transgene replacing the codons for the head domain of the viral spike. Gene Ther 2015 Mar;22(3):267-279.
van der Torren CR, Zaldumbide A, Duinkerken G, Brand-Schaaf SH, Peakman M, Stange G, Martinson L, Kroon E, Brandon EP, Pipeleers D, Roep BO. Immunogenicity of human embryonic stem cell-derived beta cells. Diabetologia 2017 Jan;60(1):126-133.
Zaldumbide A, Carlotti F, Goncalves MA, Knaan-Shanzer S, Cramer SJ, Roep BO, Wiertz EJ, Hoeben RC. Adenoviral vectors stimulate glucagon transcription in human mesenchymal stem cells expressing pancreatic transcription factors. PLoS One 2012;7 (10):e48093.
Zaldumbide A, Alkemade G, Carlotti F, Nikolic T, Abreu JR, Engelse MA, Skowera A, de Koning EJ, Peakman M, Roep BO, Hoeben RC, Wiertz EJ. Genetically engineered human islets protected from CD8-mediated autoimmune destruction in vivo. Mol Ther 2013 Aug;21(8):1592-1601
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 ‘Vascular and 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.