Retinitis pigmentosa is a collective term for a group of inherited retinal dystrophies characterized by loss of rod photoreceptors, followed by loss of cone photoreceptors. It is a disease with a... Show moreRetinitis pigmentosa is a collective term for a group of inherited retinal dystrophies characterized by loss of rod photoreceptors, followed by loss of cone photoreceptors. It is a disease with a variable clinical and genetic presentation, with regards to age at onset, severity, and disease progression. For many patients, no treatment is available at the moment, but promising advances are made in genomic medicine. In preparation for these novel therapies, this thesis focuses on the clinical characteristics and natural course of candidate genes, in order to define optimal clinical endpoints. Additionally, this thesis investigates current treatment modalities, as retinitis pigmentosa is associated with ocular comorbidities such as cataract and cystoid macular edema, which can significantly impact a patient's quality of life when left untreated. Show less
A proper immune system is essential to fight off pathogens such as viruses, bacteria, and fungi. The immune system also plays a huge role in the protection against cancer, as it can eradicate tumor... Show moreA proper immune system is essential to fight off pathogens such as viruses, bacteria, and fungi. The immune system also plays a huge role in the protection against cancer, as it can eradicate tumor cells. All immune cells are derived from hematopoietic stem cells (HSC) that undergo differentiation in a highly regulated succession of developmental steps. Each of the cell types from the immune system perform a unique specialized role, and where most of these lineages develop in the bone marrow, the T cells that make part of our adaptive immunity, develop in the thymus within a specialized environment. To achieve this, the development of each of these cell types is regulated by a variety of transcription factors.In Chapter 2 of this thesis, we reviewed the complexity of one of the important signaling pathways of hematopoietic development, the Wnt pathway. While this serves as an introduction to the fundamental research we performed, it also shines light onto potential therapeutic targets within the Wnt pathway. For further study of the Wnt pathway, we generated a novel reporter mouse, which is described in Chapter 3 of this thesis. Here we developed a reporter mouse for the Axin2 gene with the fluorescent tag mTurquoise2 with CRISPR/Cas9 genome editing tools. Based on how the genetic engineering was done to create this reporter mouse, mice that are homozygous for this reporter knock-in are also a functional knockout for Axin2. For proper functional studies, the heterozygous mice should be used.The Axin2-mTurquoise2 mouse was used in Chapter 4 of this thesis to study Wnt involvement in hematopoiesis and T cell development. We observed an increase of canonical Wnt-signaling in thymocytes from mice that have a loss of Axin2 (Axin2-TQtg/tg mice). This confirms the Wnt dosage effect that was reported previously in literature. Conclusively, these results indicate that Axin2 is required to fine-tune Wnt activity to the levels that are “just right” and cannot be maintained by Wnt activator Axin1 alone.Chapters 2, 3 and 4 focused on fundamental research on hematopoiesis and T cell development. Chapter 5 is more translational oriented and is an introductory review to thymic regenerative therapies. In Chapter 6 of this thesis, we describe the development of a combined cell and gene therapy effort to regenerate a functional thymus transplant from human Induced Pluripotent Stem Cells (iPSCs). We generated an iPSC-derived thymus by directed differentiation of human iPSCs towards thymic epithelial progenitor cells (TEPCs) using FOXN1, formation of 3-D structures from these cells which we named iPSC-derived TEPCs, or iTEPCs, and transplantation of these organoids into mice that lack a functional thymus. Functionality was demonstrated by reconstitution of functional T cells from iPSC-derived grafts, which was introduced by FOXN1 gene therapy (FOXN1 iTEPCs).Chapter 7 is the final translational research chapter of this thesis and investigated the use of iPSCs for the modeling of PIDs and the initial steps towards T cell regeneration in SCID patients. This chapter describes the iPSC generation, and its repair to use gene-corrected iPSCs from a RAG2 SCID patient to repair their disrupted immune system. The resulting iPSC model was used for disease modelling and provided novel insights into the T cell development in these RAG2-SCID patients, as we observed developmental blocks at every investigated stage of T cell development. The findings in this chapter also provide a proof-of-principle to treat a variety of SCID patients by utilizing ex vivo cell and gene therapy.Altogether, this thesis tackles two sides of the same coin: fundamentals of hematopoiesis and T cell development, and regenerative therapies for the immune system. The fundamental tools and findings in this thesis can lead to important insights to find new treatment options or improve existing therapies. Furthermore, we provide the basis for two potential therapies to treat patients with a variety of immune disorders, including DiGeorge Syndrome, SCID, age-related immune deficiencies and (post-transplant) leukemia patients that received ablative therapies. Show less
Huntington Disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in the exon 1 of the huntingtin (HTT) gene, which confers a toxic gain-of-function inducing protein... Show moreHuntington Disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in the exon 1 of the huntingtin (HTT) gene, which confers a toxic gain-of-function inducing protein aggregation and cell death. Although HD has a well-defined monogenic cause and promising HTT-lowering therapies are being tested in clinical trials, mechanism of action studies can reveal relevant information about therapeutic targets and outcomes required for successful translation into patients.One of the most advanced HTT lowering therapies for HD is a micro(mi)RNA-based gene therapy which consists of an engineered miRNA targeting the exon 1 sequence of HTT (miHTT) and delivered by adeno-associated virus (AAV) into neuronal striatal cells (AAV-miHTT).The work in this thesis describes novel mechanistic features of AAV-miHTT treatment for HD, including the targeting of toxic HTT fragments, the therapeutic spread between neuronal cells and the development of translational biomarkers to monitor its efficacy in the affected brain regions. In the light of this work, we support the reduction of HTT exon 1 fragment, the persistent efficacy in most affected brain areas, and mechanisms that improve therapeutic spread to all affected cells, as processes that potentially contribute to the successful treatment of HD patients. Show less
Biallelic CRB1 gene variations can cause retinitis pigmentosa (RP), Leber congenital amaurosis, or in some cases macular degeneration. This thesis describes the generation and analysis of RP-CRB1... Show moreBiallelic CRB1 gene variations can cause retinitis pigmentosa (RP), Leber congenital amaurosis, or in some cases macular degeneration. This thesis describes the generation and analysis of RP-CRB1 mouse and human retinas (mouse: Crb1KOCrb2LowMGCs; chapter 2. Human RP-CRB1-patient-derived organoids (chapter 4 and 5). The data indicates that the human RP-CRB1 disease can be studied in mice and human organoids. Then, we show that recombinant adeno-associated viral (rAAV)-CRB gene supplementation therapy to Müller glial cells (MGCs) of the Crb1KOCrb2LowMGCs mouse retina can protect it from stress-induced vision loss, and that human CRB2 cDNA was superior to human CRB1 cDNA (chapter 2). We then developed an improved rAAV tropism assay on human donor eyes (chapter 3). This assay shows that rAAV5 can efficiently infect Müller glial cells and photoreceptors, the target cells of a RP-CRB1 gene therapy. Also, rAAV5 infection studies outperformed rAAV9 on human retinal organoids and human donor retinas (chapter 4). Finally, we find much more early endosomes and an increase of the degradative cellular vesicles which is linked to decrease of RAB11A-postive recycling endosomes in RP-CRB1 patient organoids (chapter 5). Thus, this thesis on both human and mouse models provides new insight into retinal degeneration and rAAV gene supplementation therapies. Show less
Retinal dystrophies comprise relatively rare but devastating causes of progressive vision loss. They represent a spectrum of diseases with marked genetic and clinical heterogeneity. Mutations in... Show moreRetinal dystrophies comprise relatively rare but devastating causes of progressive vision loss. They represent a spectrum of diseases with marked genetic and clinical heterogeneity. Mutations in the same gene may lead to different diagnoses, e.g. retinitis pigmentosa or cone dystrophy. Conversely, mutations in different genes may lead to the same phenotype. The age at symptom onset, as well as the rate of vision decline, may vary widely per disease group and even within families. For most IRD cases, no effective treatment is currently available. However, preclinical studies and phase I/II/III gene therapy trials are ongoing for several IRD subtypes, and recently the first retinal gene therapy has been approved by the United States Food and Drug Administration for RPE65-associated IRDs: voretigene neparvovec-rzyl (Luxturna®). With these rapid advances in gene therapy studies, insight into the phenotypic spectrum and long-term disease course becomes crucial. The vast clinical heterogeneity presents an important challenge in the evaluation of potential efficacy in future treatment trials, and in establishing treatment candidacy criteria. This thesis responds to these challenges, providing detailed clinical descriptions of several forms of IRD that are caused by genes of interest for ongoing and future gene or cell-based therapy trials. Show less
The immune system is a complex mechanism of defence to prevent/limit infections and to guarantee homeostasis and repair. It consists of an interactive network of lymphoid organs, humoral factors... Show moreThe immune system is a complex mechanism of defence to prevent/limit infections and to guarantee homeostasis and repair. It consists of an interactive network of lymphoid organs, humoral factors and several types of specialized cells that develop from hematopoietic stem cells. In this thesis we gained additional knowledge on normal immune development, with detailed focus on the transcriptional network (Notch, Tcf1, Gata3, Bcl11b) orchestrating T-cell development.The disruption of normal lymphoid development can lead to severe illnesses known as immunodeficiencies. Severe Combined Immunodeficiency (SCID) is a devastating and fatal immune disorder affecting infants lacking a functional immune system. We focused on developing an efficient and safe gene therapy approach to correct both RAG1 and RAG2 immune defects causing SCID. The successful pre-clinical development together with a favorable safety substantiates the first phase I/II clinical trial worldwide for RAG1-SCID gene therapy.Additionally, we optimized related protocols by developing novel tools allowing better characterizing the product prior infusion. Lastly, we aimed to reduce the toxicity of the used conditioning regimens for a safer and more efficient allogeneic and autologous gene therapy transplantation outcome.Altogether, the work described in this thesis moves towards a regular enforcement of gene therapy treatment for immunodeficiencies. Show less
This thesis describes the development of microRNA-based gene therapies for ALS andSCA3. Other aspects of a successful gene therapy including the administration routes toreach the target organs and... Show moreThis thesis describes the development of microRNA-based gene therapies for ALS andSCA3. Other aspects of a successful gene therapy including the administration routes toreach the target organs and regulation of the transgene expression were also investigated.Due to the permanent nature of gene therapy, a method to modulate the transgeneexpression is desirable. Show less
By investigating the roles of CRB proteins in mouse, non-human-primate, human fetal retina, and iPSC-derived retinal organoids, this thesis describes important insights to pathobiology in CRB1... Show moreBy investigating the roles of CRB proteins in mouse, non-human-primate, human fetal retina, and iPSC-derived retinal organoids, this thesis describes important insights to pathobiology in CRB1-retinitis pigmentosa (RP) and CRB1-Leber congenital amaurosis (LCA) disease models. The thesis describes AAV gene and cell therapy-based tools as therapeutic strategies for alleviation of RP and LCA due to loss of CRB1. Show less
Huntington’s disease (HD) is a devastating neurodegenerative disease caused by a single mutation, a CAG expansion, in the huntingtin (HTT) gene. The resultant mutant HTT protein has been shown to... Show moreHuntington’s disease (HD) is a devastating neurodegenerative disease caused by a single mutation, a CAG expansion, in the huntingtin (HTT) gene. The resultant mutant HTT protein has been shown to be the predominant toxic entity in the HD pathogenesis and therapeutic strategies that aim to lower the mutant HTT show a great promise. The main objective of this work is to demonstrate a preclinical efficacy of an adeno-associated virus (AAV)-delivery of micro (mi)RNA-based gene therapy for the treatment of HD. We have tested various therapeutic miRNAs to achieve overall HTT protein lowering in HD rodent models and induced pluripotent stem cell (iPSC)-derived HD patient neuronal cultures. Excitingly, we have demonstrated HTT lowering by the AAV5-miHTT in all HD models tested so far with no undesired events, which strongly supported the continuation of preclinical testing in large animals. Furthermore, we provided an evidence suggesting that therapeutic miRNAs can be also active in the nucleus, extending their range of applicability. The possibility to use exosome-enriched vesicles as carriers of pharmacokinetic/pharmacodynamic (PK/PD) measures for the AAV5-miHTT gene therapy, that would signal the presence of the active therapeutic miRNAs in the brain, was further explored in preparation for a first clinical trial in humans. Show less
Oncolytic viruses are highly promising agents for cancer therapy. Reovirus type 3 Dearing and adenovirus type 5 are potent representatives of this new type of treatment. They have been tested... Show moreOncolytic viruses are highly promising agents for cancer therapy. Reovirus type 3 Dearing and adenovirus type 5 are potent representatives of this new type of treatment. They have been tested in a multitude of clinical trials. In these studies reovirus and adenovirus display a good safety profile, but anti-tumour efficacy should be improved. The work described in this thesis mainly concentrated on improving the entry and spread of reoviruses and adenoviruses in tumours by means of genetic modification of the viruses. Show less
Despite major microsurgical improvements the clinical outcome of peripheral nerve surgery is still regarded as suboptimal. Over the past decade several innovative techniques have been... Show more Despite major microsurgical improvements the clinical outcome of peripheral nerve surgery is still regarded as suboptimal. Over the past decade several innovative techniques have been developed to extend the armamentarium of the nerve surgeon. This thesis evaluates the potential of gene therapy in the context of peripheral nerve repair. The overall goal of the work presented in this thesis is to enhance peripheral nerve regen- eration by stimulating axonal growth and reducing misdirection. We set out to achieve this by increasing the therapeutic potential of Schwann cells through gene therapy. Show less
Glioblastoma multiforme (GBM) is the most malignant variant of glioma. This tumor does not only display an extremely aggressive, invasive growth pattern, but is also very difficult to treat. With a... Show moreGlioblastoma multiforme (GBM) is the most malignant variant of glioma. This tumor does not only display an extremely aggressive, invasive growth pattern, but is also very difficult to treat. With a two-year survival rate of 40% and a median survival of 12-18 months after treatment, prognosis is poor. Current treatment options are not successful in halting tumor progression and GBM tumors are highly heterogeneous, display all kinds of anti-apoptotic escape routes, suppress the immune system, invade the surrounding parenchyma with unmatched aggressiveness and possess a whole array of tools to rearrange the extra tumoral environment to their advantage. The aim of this thesis is to combine the strengths of gene-therapy and bioluminescence Imaging (BLI) for the development of novel reporter systems in order to study glioma tumor biology and its response to therapeutic compounds. We optimized the currently available BLI luciferases (Gaussia luciferase, Vargula hilgendorfi) and assays (Gluc blood assay, Mycoplasma detection assay). We explored a new multimodal targeted liposome formulation with increased relaxivity for the treatment and imaging of cancer. Finally we combined the newly developed and enhanced reporters to test a new therapeutic combination for the treatment of Glioma (TRAIL, Lanatoside C). Show less
Misdirection of regenerating motor axons is one of the factors that can explain the disappointing recovery of function often observed after nerve injury and repair. In the first part of this thesis... Show moreMisdirection of regenerating motor axons is one of the factors that can explain the disappointing recovery of function often observed after nerve injury and repair. In the first part of this thesis we quantified misdirection of motor axon regeneration after different types of nerve injury and repair in the rat sciatic nerve model, and we investigated the impact on functional recovery with ankle motion analysis. In the second part of this thesis we investigated two tools to guide regenerating motor axons in vivo: mechanical guidance with multichannel nerve tubes and biological guidance with gene therapy. Show less
Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, caused by mutations in the DMD gene that prevent synthesis of dystrophin. Fibers that lack dystrophin are sensitive... Show moreDuchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, caused by mutations in the DMD gene that prevent synthesis of dystrophin. Fibers that lack dystrophin are sensitive to exercise-induced damage, resulting in progressive muscle wasting, loss of ambulation and premature death. There is no cure, but several therapeutic approaches are clinically tested. At best, these clinical interventions result in the expression of low dystrophin levels. Fortunately, expression of wild type levels is not needed, as both humans and mice expressing ~50% of dystrophin do not show pathology. Detailed studies on which dystrophin levels are needed to prevent pathology and improve muscle function have been performed in this thesis. After the set-up of good outcome measures and serum biomarkers to monitor disease progression, two new innovative mouse models expressing low levels of dystrophin based on skewed X-inactivation were generated. In the mdx-Xist__hs model we observed that <15% dystrophin already improved muscle performance, while histopathology was largely with >15% dystrophin. To protect muscles from exercise-induced damage >22% dystrophin was needed. Dystrophin levels between 3-21% prevent the development of dilated cardiomyopathy in 10 months old mice. Mice lacking both dystrophin and its homologue utrophin, mimic the human phenotype and die before the age of 12 weeks. In these mice, <10% dystrophin improved life expectancy and muscle function while >10% dystrophin was needed to improve histopathology. These findings are encouraging for ongoing and future clinical trails. Show less
Recombinant viral vectors hold great promise in the field of cancer gene therapy. While a plethora of viruses is being evaluated as oncolytic agents, human adenoviruses of serotype 5 (HAdV-5) are... Show moreRecombinant viral vectors hold great promise in the field of cancer gene therapy. While a plethora of viruses is being evaluated as oncolytic agents, human adenoviruses of serotype 5 (HAdV-5) are among the most popular of viruses to be developed. Although clinical studies have demonstrated safety of cancer gene therapy with HAdV-5-derived vectors, the efficacy still needs further enhancement. Several factors have been identified that limit the anti-tumor efficacy. One major bottleneck is the inadequate penetration and spread of the virus within the tumor. This is attributable, at least in part, to the low or heterogeneous expression of the coxsackie and adenovirus receptor (CAR) on the tumor cells. This thesis describes the development and preclinical evaluation of novel tumor-targeted HAdV-5 vectors, through implementing the genetic fusion of capsid proteins (protein IX and fiber) with a variety of tumor-targetin g polypeptides. Show less
Approximately one million total hip replacement operations are performed worldwide annually, mostly for osteoarthritis and rheumatoid arthritis. A major complication in total hip arthroplasties is... Show moreApproximately one million total hip replacement operations are performed worldwide annually, mostly for osteoarthritis and rheumatoid arthritis. A major complication in total hip arthroplasties is loosening of the prosthesis leading to pain and walking difficulties and a higher risk for dislocations and pathological fractures.11 Within ten years of primary hip replacement 7-13 percent of patients need revision surgery due to loosening of the implant.16 The number of revision surgeries in elderly patients is likely to increase considerably in the next decades, due to the tendency to insert orthopaedic implants at younger ages and the longer life expectancy of patients. Revision surgery has a high complication rate in elderly patients1,20,23,25 and is associated with less improvement in social outcome, compared to primary hip arthroplasty in patients of all ages.24 Many studies have focused on the relatively good technical outcome of revision hip arthroplasty in patients of all ages. However, the impact on the patient__s life, especially in elderly patients has been underemphasised. This thesis was set up to develop and analyse an alternative treatment for hip prosthesis loosening. We used a gene-directed enzyme-prodrug therapy to kill and remove interface tissue, after which we injected bone cement around the prosthesis percutaneously under CT- and fluoroscopy guidance. Show less
The studies described in this thesis focus on gene therapeutic strategies to target pathological vascular wall remodeling after PT(C)A or bypass surgery. Inflammatory processes and extracellular... Show moreThe studies described in this thesis focus on gene therapeutic strategies to target pathological vascular wall remodeling after PT(C)A or bypass surgery. Inflammatory processes and extracellular proteases, both activated by mechanical and vascular injury caused by these interventions, are thought to contribute largely to the development of post-angioplasty restenosis and vein graft disease. Therefore, viral and non-viral gene therapy techniques were used in these studies to deliver genes encoding protective as well as inhibiting proteins in order to modulate the inflammatory cascade (i.e. IL-10 and the MCP-1/CCR-2 pathway) in the first part of this thesis and the plasminogen activator and MMP-system in the second part. Finally, the expression of several involving genes was blocked locally by RNA interference techniques in the last part of this thesis. The possibilities and effects of these gene therapy applications were studied in cell cultures, in a human saphenous vein organ culture model and in two mouse models of restenosis and vein graft disease. Altogether, these studies provided more insight into the pathophysiology of post-interventional remodeling and several potential therapeutic strategies were assessed. Show less
The research described in this thesis consist of 2 parts: the first part involves studies on the influence of chemokines in cardiovascular disease. Chemokines are inflammatory proteins which play a... Show moreThe research described in this thesis consist of 2 parts: the first part involves studies on the influence of chemokines in cardiovascular disease. Chemokines are inflammatory proteins which play a pivotal role in atherosclerosis and myocardial ischemia. We identify 3 chemokines (CCL3, CCL5 and CCL18) whose levels are not only elevated during myocardial ischemia, but are also predictive of future cardiovascular events. Further studies focus on the individual role of CCL18 as well as CCL3 in atherogenesis and atherosclerotic plaque destabilization. The first is seen to recruit T-lymphocytes and the latter neutrophil granulocytes into the plaque, possibly augmenting plaque growth and destabilization. The second part focuses on the effect of gene modulation on vascular function. It start of with a study on the influence of aging in our atherosclerotic plaque mouse model. Additional genetic microarray revealed the Quaking gene as a possible modulator of atherosclerosis. This observation is further explored in studies which show a link between Quaking genetic polymorphisms and an enhanced risk of developing in-stent restenosis following percutaneous coronary intervention. This is partly mediated by disturbed vascular smooth cell function. Finally, the MEF2 gene is studied for its role in myocardial infarction as genetic mutations in the MEF2A gene are associated with enhanced risk for a myocardial infarction. In a mouse model, we show that this is primarily due to decreased endothelial cell function, leading to plaque erosion. Show less
While currently available therapeutic options for the treatment of acute myocardial infarction are sufficient for the treatment of symptoms, the underlying causes usually remain unresolved, being... Show moreWhile currently available therapeutic options for the treatment of acute myocardial infarction are sufficient for the treatment of symptoms, the underlying causes usually remain unresolved, being loss of electrically active, contractile, myocardial tissue. Recently, extensive research has been performed in the field of cell and gene therapy. The ultimate aim of these therapies is to __heal__ the infarcted area on a more biological basis, by repopulating the damaged area with __new__ cells that contribute to proper cardiac function, including electrical activation of the myocardium. In order to comprehend the potential therapeutic value and hazard of cell modification and transplantation for ischemic heart diseases, one should consider the heart as a highly integrative, electromechanical organ. Therefore, the aim of this thesis was to explore, from a mechanistic and electrophysiological point of view, the integrative and functional aspects of cell modification and transplantation as therapeutic options to cure the damaged, ischemic heart. Show less
Adoptive transfer of T cell immunity has been proposed as an attractive form of cancer immunotherapy in cases where the endogenous T cell repertoire is immune tolerant. In this thesis murine models... Show moreAdoptive transfer of T cell immunity has been proposed as an attractive form of cancer immunotherapy in cases where the endogenous T cell repertoire is immune tolerant. In this thesis murine models are used to study two different approaches to generate T cell grafts with defined tumor specificities. First the feasibility of minor antigen specific T cell depletion to limit the development of Graft-versus-Host Disease after allogeneic stem cell transplantation in combination with donor lymphocyte infusions is addressed. In the second part T cell receptor gene transfer is used to generate tumor specific T cells. Show less
