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
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
