Mutations in the CRB1 gene can cause retinal dystrophies such as Retinitis Pigmentosa or Leber Congenital Amaurosis . These patients experience progressive vision loss which ultimately leads to... Show moreMutations in the CRB1 gene can cause retinal dystrophies such as Retinitis Pigmentosa or Leber Congenital Amaurosis . These patients experience progressive vision loss which ultimately leads to blindness. Currently, there are no treatment options available for these patients.This thesis provides novel information on AAV.hCRB gene augmentation therapy in CRB1 mutant animal and human-derived models. We show the phenotype of (1) a novel mouse model with CRB2 ablation specifically in rod photoreceptor cells with loss of retinal function (Chapter 2), (2) a Crb1 mutant brown Norway rat with severe and early onset progressive vision loss (Chapter 3), (3) CRB1 patient-derived retinal organoids (Chapter 4), and (4) CRB1KO and CRB1KOCRB2+/- LCA-like retinal organoids (Chapter 5). Next, AAV-mediated gene augmentation was explored in Crb1 mutant rats (Chapter 3) and CRB1 patient-derived and CRB1KO LCA retinal organoids (Chapter 4 and 5). Finally, single-cell RNA sequencing was performed on AAV.hCRB treated CRB1 patient-derived retinal organoids (Chapter 4). To our knowledge this is the first time that an improved phenotype after AAV.hCRB gene augmentation in CRB1 RP patient-derived and CRB1KO LCA retinal organoids is observed, providing essential information for future gene therapy possibilities in patients with a mutation in the CRB1 gene. Show less
Familial hypercholesterolemia (FH) is a genetic disorder characterized by high levels of low density lipoprotein cholesterol (LDL-C) and increasing the risk of cardio vascular diseases. FH and many... Show moreFamilial hypercholesterolemia (FH) is a genetic disorder characterized by high levels of low density lipoprotein cholesterol (LDL-C) and increasing the risk of cardio vascular diseases. FH and many other liver diseases can possibly be treated with RNA interference (RNAi). RNAi is a natural process of regulation of gene expression by binding of small RNA molecules to complementary sequences in the mRNA of a gene and hence inducing its degradation or translational repression. In this thesis, we aimed at developing a safe and robust RNAi-based therapy for FH by inhibiting Apolipoprotein B100 (ApoB). ApoB is a primary component of the low density lipoprotein (LDL), a lipoprotein that transports __bad__ cholesterol LDL-C. We have shown that RNAi therapy using AAV-delivered artificial miRNA is a promising approach for treatment of FH and possibly other liver diseases. Our mechanistic studies revealed differences in shRNA and miRNA processing and functioning in vivo. Finally, our findings have significant impact on understanding and overcoming toxicity and off-targeting related problems of RNAi based gene therapy in the liver using AAV vectors. Show less
The overall aim of this thesis is to contribute to the engineering of more selective and effective oncolytic Adenovirus (Ad) vectors. Two general approaches are taken for this purpose: (i) genetic... Show moreThe overall aim of this thesis is to contribute to the engineering of more selective and effective oncolytic Adenovirus (Ad) vectors. Two general approaches are taken for this purpose: (i) genetic capsid modification to achieve Ad retargeting (Chapters 2 to 4), and (ii) directed evolution to improve the cytolytic potency of Ad (Chapter 5). In order to provide some context for these approaches, Chapter 1, part II gives a brief background on Ad biology and vectorology. Further, in Chapter 1, part III, a broad overview is provided of the ways that evolution-based engineering has previously been used to generate or improve viral vectors. Chapters 2 and 3 focus on the modification of the minor Ad capsid protein IX (pIX). pIX is present on the faces of the Ad capsid icosahedron, functioning as __cement__ between the much larger hexon proteins. Previously, the C-terminus of pIX proved serviceable as an anchor for the genetic capsid incorporation of targeting ligands and other heterologous moieties. In Chapter 2, a new system is described that allows for the rapid functional testing of new pIX-ligand fusion proteins. In this system, lentiviral vectors are used to generate cells stably expressing the pIX variant of interest. Large-scale infection on such cells with a pIX-deleted Ad vector subsequently yields an Ad vector preparation phenotypically pseudotyped with the new pIX variant. This system thus allows rapid analysis of new pIX-ligand fusions in the context of the Ad capsid without having to genetically modify the Ad genome. In Chapter 3, the lentiviral vector-based pIX-pseudotyping system is put to use for the analysis of a new pIX fusion protein harboring a single-chain T-cell receptor (scTCR) as a targeting ligand. The concerning scTCR was directed against the intracellular cancer-testis antigen melanoma-associated antigen-A1. Importantly, this chimeric pIX molecule proved to be efficiently incorporated into the Ad capsid. Moreover, Ad transduction studies showed evidence of the capsid-displayed scTCR to mediate a degree of specific target cell transduction via the cognate peptide-MHC complex. Analogously as done for pIX, Chapter 4 describes a phenotypical pseudotyping approach for fiber. The Ad-encoded fiber protein is present as a trimeric rod-like structure that extends from the vertices of the Ad capsid icosahedron. Its outward-facing, C-terminal __knob__ domain is responsible for binding the Coxsackie and adenovirus receptor (CAR), Ad__s in vitro primary cell surface attachment protein. With its prominent role in native receptor binding, the Ad fiber is logically subject to many capsid modification strategies that aim at altering Ad tropism. Thus to facilitate expedited testing of new fiber variants, a lentiviral vector-based, fiber-pseudotyping system was set up. This involved optimization of the fiber (variant) expression cassettes by inclusion of the tripartite leader sequence of Ad__s major late transcription unit. A second objective of this study was to functionally assess a new chimeric fiber harboring a tumor antigen-directed single-chain variable fragment (scFv) antibody. Although this fiber variant showed some degree of target binding and formed stable trimers, it displayed problems regarding capsid incorporation ability, functionality within the capsid, and folding of its scFv constituent. Thus, this particular fiber proved not suitable for Ad retargeting. Finally, Chapter 5 describes the development and validation of a novel evolution-based engineering approach for Ad. To date, most Ad-based vectors have been generated through molecular design. Although this rational tailoring of Ad has led to significant vector improvements, it is often still hampered by our limited understanding of the intricate viral function-structure relationships. Therefore, __random__ virus engineering strategies (see Chapter 1, part III) may be a useful alternative or complementary approach for the generation of new or improved viral vectors. In this regard, the high mutation rates of RNA viruses have proven readily exploitable in adaptation studies to achieve vectorological goals. Thus, it was hypothesized that a mutator Ad polymerase-based, __accelerated evolution__ procedure would likewise be of use for Ad vector engineering. To develop such a system, the intrinsic mutation rate of Ad replication was sought to be increased by modification of the Ad-encoded DNA polymerase (Ad pol). This was done by mutation of residues within regions putatively important for nucleotide selection or proofreading. A mutation-accumulation and deep sequencing strategy was subsequently used to identify any mutators among the Ad pol mutants. Finally, the mutator polymerase-based directed evolution approach was validated by conducting an evolution procedure aimed at increasing Ad__s oncolytic potency, and by subsequent characterization of resultant bioselected virus populations and isolated clones. Show less
In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the... Show moreIn this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the fact that many for gene therapy interesting cell types do not, or only at low levels express the CAR receptor, necessary for infection. We developed a linker protein consisting of the virus-binding moiety of CAR genetically fused to the chicken protein avidin. Biotinylated ligands for cell specific receptors are bound to the linker protein via the avidin-biotin interaction. This now targeting protein is used to redirect adenovirus vectors to previously refractory cell types. Using this technology endothelial cell lines as well as primary endothelial cells can by infected at low MOI__s using an biotinylated cyclic RGD peptide. Primary bone marrow derived macrophages and macrophage cell lines are easily infected using a biotinylated dA6dG10 oligo nucleotide ligand. In vivo experiments showed a marked reduction of adenovirus mediated transgene expression by the liver, the organ responsible for virus uptake when unmodified adenovirus vectors are administered, after addition of several different ligands to the virus via the linker protein. Show less
