The work in our program is mainly based on the multifaceted use of virus and stem-cell expertise.
In the DNA editing field we have made major steps to vectorise the targeted nucleases that are required for efficient DNA editing. A hallmark paper Goncalves and collaborators describes the differential stability of TALE nucleases in adenovirus and lentivirus vectors (Holkers et al., 2013). The adenovirus vectors delivered the TALE nuclease genes much more faithful than lentivirus vectors. This was built upon by showing the efficient targeted gene replacement using adenovirus vector mediated transfer of the CRISPR guides, the cas9 genes, and the donor sequences that should become integrated into the host cell genome (Holkers et al., 2014). That this powerful technology not only works in cell lines but also in human embryonal and induced pluripotent stem cells was demonstrated in a recent study by Mikkers and Goncalves (Chen et al., 2017). While much of this work was geared up for use in Duchenne muscular dystrophy, this work has been the stepping stone on which we built the consortium that aims at modifying the genome in human hematopoietic stem cells (hHSC). It goes without saying that if we demonstrate the feasibility of this approach in hHSC, many more single-gene defects that affect the blood-cell compartment are amendable to gene-editing based therapeutic interventions (i.e. immune deficiencies, thalassemia’s).
Our immune-evasion program (Zaldumbide, Ressing) aims at protecting cells from the destructive action of the immune system. Initially Zaldumbide and Hoeben pioneered the immune-stealth technology, with which we can prevent the proteasomal degradation of specific proteins and therefore the presentation of their peptides in class I molecules. In a next step Hoeben and Zaldumbide, in collaboration with labs of Roep, De Koning, and Wiertz (UMC, Utrecht) designed a method to mask entire cells by expressing a herpesvirus gene that encodes an inhibitor of the TAP transporters. This inhibition blocks peptide translocation of the peptides into the ER and their loading on class I molecules and we showed that this can protect the insulin-producing β-cells from the destructive action of insulin-specific T cells (Zaldumbide et al., 2013). For this project Zaldumbide optimized the protocols for gene transfer into human β-cells. These methods and vector are also key in the joint research project with Carlotti and De Koning (Nephrology) in which we showed that isolated human β-cells frequent convert to α cells (Spijker at al., 2013). Ressing identified the EBV gp150 as a protein with immune-evasive activity (Gram et al., 2016). This protein is heavily glycosylated and generates a glycan shield on the EBV-infected cells that thwarts antigen presentation by class I, class II, and CD1d. In this project we again collaborated with Wiertz.
Our collaboration with the Roep and De Koning labs also led Zaldumbide to identify a new highly immunogenic peptide derived from the insulin gene. The peptide is derived from a Defective Ribosomal Product (DRIP), and is target of T-cells circulating is some Type I diabetes patients. These T-cells are capable of killing human β cells and thereby may be diabetogenic (Kracht et al., 2017). This is a highly relevant observation with clinical implications.
Viruses are also key in our oncolytic virus project. Reoviruses are oncolytic viruses par excellence owing to their intrinsic preference for infecting and killing tumour cells. While the use of wildtype reovirus as an oncolytic agent demonstrated the safety and potential efficacy of the approach, there are significant limitations to the use of wt reoviruses. One of these is the downregulation of the reovirus receptor on several tumour types. This prompted Hoeben and collaborators to isolate reovirus mutants that bypass the receptor dependency. The resulting jin mutants now also infect cells lacking the JAM-A receptor (Van den Wollenberg et al., 2012). These jin-mutants also allowed our group to generate the world’s first oncolytic reoviruses that stably express a transgene (Van den Wollenberg et al., 2015). Our reoviruses have been selected by the Dutch OVIT consortium as a prime candidate for clinical development and are now tested in prostate, pancreatic, bladder, and brain cancer in in-vitro studies in cell cultures, in murine animal models, as well as in viable human tumour slices.
Understanding the molecular and biochemical cues such as TGFβ signalling that govern (cardiovascular) cell differentiation and behaviour is used to improve cardiovascular function. Injection of cardiac progenitor cells into the infarct border zone revealed a major contribution of exosomes to the cells beneficial effects (Vrijsen 2016; Maring 2018). To unravel the role of (endogenous) epicardial cells in heart development and repair we developed an in vitro cell system (Moerkamp 2016; Dronkers 2018) and are currently using this model to identify EMT activators. Rare genetic disorders like PAH, HHT and FOP, show disturbed TGFβ signalling and enhanced endothelial to mesenchymal transition thereby impairing cardiovascular cell differentiation and function of the cardiovascular system. We aim to understand the molecular and physiological mechanisms behind these diseases and employ this knowledge to identify e.g. small molecules (Sánchez-Duffhues 2018) to steer the differentiation of cardiovascular cells in vitro and in vivo. Furthermore, using e.g. growth-factor cocktails, exosomes, DPP-4 inhibitors (Dingenouts 2017) and transcription factors, we explore the capacity of (stem) cells to home to sites of injury and/or repair the (cardiovascular) tissue from within.
In parallel we use large cohorts of both healthy and affected people to identify markers of disease progression in type 2 diabetes. In these studies we combine longitudinally collected, highly standardized, clinical data with repeated sampling of biomaterials. Various data integration methods are used and or further developed. This has facilitated extensive research into the potential of various omics measurements in stratification of patients according their risk for treatment failure, progression of diabetes and development of diabetic complications. We have for instance shown that several gene variants impact on response to metformin or DPP-4 inhibitor treatment in type 2 diabetes patients (‘t Hart et al., 2013). In addition we investigate gene activity in blood cells (transcriptomics, Slieker et al., 2018), metabolomics (‘t Hart et al, 2018), lipidomics and peptidomics in relation to diabetes type 2 progression.